Analyte sensors featuring enhancements for decreasing interferent signal

ABSTRACT

Analyte sensors are being increasingly employed for monitoring various analytes in vivo. Analyte sensors may feature enhancements to address signals obtained from interferent species. Some analyte sensors may comprise a working electrode comprising an active area disposed thereon and electrode asperities laser planed therefrom. Some analyte sensors may comprise an interferent-reactant species incorporated therewith. Some analyte sensors may comprise an interferent scrubbing electrode. Combinations of these enhancements may additionally be employed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalApplication No. 63/049,210, filed Jul. 8, 2020, which is incorporated byreference herein in its entirety.

BACKGROUND

The detection of various analytes within an individual can sometimes bevital for monitoring the condition of their health. Deviation fromnormal analyte levels can often be indicative of a number ofphysiological conditions. Glucose levels, for example, can beparticularly important to detect and monitor in diabetic individuals. Bymonitoring glucose levels with sufficient regularity, a diabeticindividual may be able to take corrective action (e.g., by injectinginsulin to lower glucose levels or by eating to raise glucose levels)before significant physiological harm occurs. Monitoring of otheranalytes may be desirable for other various physiological conditions.Monitoring of multiple analytes may also be desirable in some instances,particularly for comorbid conditions resulting in simultaneousdysregulation of two or more analytes in combination with one another.

Many analytes represent intriguing targets for physiological analyses,provided that a suitable detection chemistry can be identified. To thisend, in vivo analyte sensors configured for assaying variousphysiological analytes have been developed and refined over recentyears, many of which utilize enzyme-based detection strategies tofacilitate detection specificity. Indeed, in vivo analyte sensorsutilizing a glucose-responsive enzyme for monitoring blood glucoselevels are now in common use among diabetic individuals. In vivo analytesensors for other analytes are in various stages of development,including in vivo analyte sensors capable of monitoring multipleanalytes. Poor sensitivity for low-abundance analytes may be especiallyproblematic for some analyte sensors, particularly due to backgroundsignal arising from interaction of an interferent with a workingelectrode or other analyte sensing chemistry components.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures are included to illustrate certain aspects of thepresent disclosure, and should not be viewed as exclusive embodiments.The subject matter disclosed is capable of considerable modifications,alterations, combinations, and equivalents in form and function, withoutdeparting from the scope of this disclosure.

FIG. 1 shows a diagram of an illustrative sensing system that mayincorporate an analyte sensor of the present disclosure.

FIGS. 2A-2C show cross-sectional diagrams of analyte sensors comprisinga single active area.

FIGS. 3A-3C show cross-sectional diagrams of analyte sensors comprisingtwo active areas.

FIG. 4 shows a cross-sectional diagram of an analyte sensor comprisingtwo working electrodes, each having an active area present thereon.

FIG. 5 is a diagram showing a top view of a conventional carbon workingelectrode having an active area thereon.

FIG. 6A shows a photograph of a top view of a working electrode havingno membrane disposed thereon. FIG. 6B is a depth profile along the lineindicated in FIG. 6A.

FIG. 7A shows a photograph of a top view of a working electrode having amembrane disposed thereon. FIG. 7B is a depth profile along the lineindicated in FIG. 7A.

FIG. 8 is a photograph showing a 3D view of a laser planed workingelectrode, in accordance with one or more aspects of the presentdisclosure.

FIG. 9A is a depiction of a convention sensor having no incorporatedinterferent-reactive species. FIG. 9B is a depiction of the sensor ofFIG. 9B incorporating interferent-reactive species, in accordance withone or more aspects of the present disclosure.

FIG. 10 is a depiction of a sensor electrode configuration comprising ascrubbing electrode, in accordance with one or more aspects of thepresent disclosure.

FIG. 11 is a depiction of a sensor electrode configuration comprising apermeable scrubbing electrode, in accordance with one or more aspects ofthe present disclosure.

FIG. 12 is a depiction of a sensor electrode configuration comprising anon-permeable scrubbing electrode and a permeable scrubbing electrode,in accordance with one or more aspects of the present disclosure.

FIG. 13A shows a photograph of a top view of a working electrode havingno membrane and no active area disposed thereon. FIG. 13B is a depthprofile along the line indicated in FIG. 13A. FIG. 13C shows aphotograph of a top view of the working electrode of FIG. 13A afterlaser planing, in accordance with one or more aspects of the presentdisclosure. FIG. 13D is a depth profile along the line indicated in FIG.13C.

FIG. 14A shows a photograph of a top view of a working electrode havingno membrane and an active area disposed thereon after laser planing, inaccordance with one or more aspects of the present disclosure. FIG. 14Bis a depth profile along the line indicated in FIG. 14A.

FIG. 15 is a graph of a paired-difference test comparing planed andunplaned working electrodes having either an active area or lacking anactive area in response to the interferent ascorbic acid.

FIGS. 16A-16E show photographs of working electrodes. FIGS. 16A and 16Care not laser planed. FIGS. 16B, 16D, and 16E are laser planed, inaccordance with one or more aspects of the present disclosure.

FIG. 17 is a sensor configuration for inclusion of aninterferent-reactant species layer, according to one or more embodimentsof the present disclosure.

FIG. 18 is an ascorbic acid calibration curve for analyte sensors ofFIG. 17 comprising an interferent-reactant species layer, according toone or more aspects of the present disclosure.

FIG. 19 is a glucose calibration curve for analyte sensors of FIG. 17comprising an interferent-reactant species layer, according to one ormore aspects of the present disclosure.

FIGS. 20, 21A, 21B, and 22 are sensor current traces of sensorscomprising scrubbing electrodes, in accordance with one or more aspectsof the present disclosure.

FIG. 23 is a sensor configuration for inclusion of a permeable scrubbingelectrode, according to one or more aspects of the present disclosure.

FIG. 24 is a sensor current trace of the sensor of FIG. 24, inaccordance with one or more aspects of the present disclosure.

DETAILED DESCRIPTION

The present disclosure generally describes analyte sensors suitable forin vivo use and, more specifically, analyte sensors featuring one ormore enhancements for reducing or eliminating signals indicative ofinterferent species to promote improved detection sensitivity, andmethods for production and use thereof.

Such enhancements may include decreasing the availability of a workingelectrode surface upon a sensor tail (the portion of a sensor forinsertion into a tissue), particularly the availability of a carbonworking electrode upon a sensor tail upon which interferents may reactand contribute to signal not associated with the analyte. Othercomponents of an analyte sensor may also react with an interferent andcontribute to the signal at the carbon working electrode. Aspects of thepresent disclosure include, alone or in combination, planing asperitiesfrom a carbon working electrode, including compounds that react withinterferents to prevent their interaction with a carbon workingelectrode, and/or addition of a scrubbing electrode to react withinterferents to prevent their interaction with a carbon workingelectrode. In one or more aspects, the enhancements described herein maydecrease the sensitivity of the sensor to interferents (e.g., byprohibiting or reducing interferents from generating signal at theworking electrode, such as by eliminating excess carbon electrodesurface using sensing chemistries and/or membranes) and/or decrease thelocal concentration of interferents at the working electrode (e.g., by“pre-reacting” the interferents such that they do not or substantiallydo not reach the working electrode). While not necessary, when thesignal of the analyte of interest is not compromised, one or all of theenhancements described herein may be used in combination with a workingelectrode having a low working potential below the oxidation potentialof the interferents of interest. In some instances, analyte sensorsincorporating a low potential working electrode may further incorporatea low potential redox mediator to enhance detection of the analytesignal of interest of such low working potentials.

Generally, and without limitation, the embodiments of the presentdisclosure comprising one or more interferent enhancements may permit atleast a reduction in interferent signal, such as an ascorbic acidinterferent signal, compared to a sensor lacking said enhancement(s), inthe range of greater than about 20%, which may be up to 100%, or such asin the range of about 20% to about 70% or greater, and preferably atleast about 40% greater, at least about 45% greater, or at least about50%, encompassing any value and subset therebetween and in which theupper and lower limits are separable, as detailed herein. The amount ofinterferent reduction may depend on a number of factors including, butnot limited to, the particular configuration of the sensor (e.g., whichone or more enhancements are selected), the concentration of theinterferent within a bodily fluid, and the like, and any combinationthereof.

The analyte sensors described herein comprise a sensor tail comprisingat least one working electrode, particularly a carbon working electrode,and an active area disposed thereupon. A mass transport limitingmembrane is then disposed upon the carbon working electrode (i.e.,disposed upon both the active area and any extraneous carbon workingelectrode lacking the active area forming the sensor tail).

Various carbon electrode asperities may exist along the edges of thecarbon working electrode, where they may be insufficiently coated or arenot coated at all with the mass transport limiting membrane, therebyproviding a carbon surface for interferents to undergo a reaction andcontribute to the measured signal at the working electrode. As usedherein, the term “asperity,” and grammatical variants thereof, refers toa rough edge along a surface (e.g., along a working electrode).Asperities may be in the form of a ridge along the edge of a workingelectrode, thereby leading to insufficient coating of a mass transportlimiting membrane in this location. To reduce or eliminate suchinterferent signals, the present disclosure provides for planing of oneor more edges of the carbon working electrode to remove carbonasperities therefrom, thereby affording a more uniform profile of theworking electrode surface. Where the working electrode is formed from amaterial other than carbon, such asperities may be equally present inthe composition of the particular working electrode (“electrodeasperities”).

Separate or in combination with planing one or more edges of the carbonworking electrode to remove carbon asperities, the present disclosurefurther provides analyte sensors comprising one or more means to preventor reduce an interferent's access to the working electrode. Inparticular, one or more enzymatic or chemical compounds may beincorporated into the analyte sensor which reacts with the interferentof interest to render it inactive such that it cannot contribute to themeasured signal at the working electrode. Alternatively, or again incombination, a scrubbing electrode may be incorporated into the analytesensor which reacts with the interferent of interest to render itinactive such that it cannot contribute to the measured signal at theworking electrode.

Particular details and further advantages of each type of enhancementare described in further detail herein. Depending on particular needs,the analyte sensors of the present disclosure may be configured todetect one analyte or multiple analytes simultaneously or nearsimultaneously.

Analyte sensors employing enzyme-based detection are commonly used forassaying a single analyte, such as glucose, due to the frequentspecificity of enzymes for a particular substrate or class of substrate.Analyte sensors employing both single enzymes and enzyme systemscomprising multiple enzymes acting in concert may be used for thispurpose. As used herein, the term “in concert,” and grammatical variantsthereof, refers to a coupled enzymatic reaction, in which the product ofa first enzymatic reaction becomes the substrate for a second enzymaticreaction, and the second enzymatic reaction or a subsequent enzymaticreaction serves as the basis for measuring the concentration of ananalyte. Moreover, a combination of enzymes and/or enzyme systems may beemployed to detect more than one analyte type. Using an in vivo analytesensor featuring an enzyme or enzyme system to promote detection may beparticularly advantageous to avoid the frequent withdrawal of bodilyfluid that otherwise may be required for analyte monitoring to takeplace.

In vivo analyte sensors monitor one or more analytes in a biologicalfluid of interest such as dermal fluid, interstitial fluid, plasma,blood, lymph, synovial fluid, cerebrospinal fluid, saliva,bronchoalveolar lavage, amniotic fluid, or the like. Such fluids maycomprise one or more interferents that can react with the workingelectrode of the analyte sensor, either directly on the workingelectrode itself (e.g., carbon working electrode) or with one or moresensing chemistry components disposed thereupon (e.g., the redox polymerdescribed hereinbelow). As used herein, the term “interferent,” andgrammatical variants thereof, refers to any electroactive speciespresent that are not an analyte(s) of interest (e.g., in vivoelectroactive species that are not an analyte(s) of interest) within abodily fluid (e.g., interstitial fluid, and the like). Examples include,but are not limited to, ascorbic acid (vitamin C and also referred to asascorbate), glutathione, uric acid, paracetamol (acetaminophen),isoniazid, salicylate, and the like, and any combination thereof. Thereaction of these interferents with the working electrode can create anelectrochemical signal that is inseparable or not easily separable fromsignal originating from the analyte of interest, which may complicatethe accurate detection of such analytes, particularly those inlow-abundance (e.g., low-to sub-millimolar concentrations). Theelectrochemical signal generated by an interferent may be particularlyproblematic as the signal from the interferent becomes closer inmagnitude to that of the signal from the target analyte. This may occur,for example, when the concentration of the interferent approaches orexceeds the concentration of the analyte of interest. Some interferentsare ubiquitous in vivo and are not easily avoided. Therefore, techniquesto minimize their influence during in vivo analyses may be highlydesirable.

The present disclosure provides analyte sensor enhancements that, eitheralone or in combination with other enhancements, may improve detectionsensitivity for both single analytes and multiple analytes incombination with one another, as explained in further detailhereinbelow. Namely, the present disclosure provides analyte sensorshaving reduced carbon working electrode edge asperities and/orincorporated compounds or scrubbing electrodes that may afford decreasedbackground signal resulting from in vivo interferents. Although certainaspects of the present disclosure are directed to enhancement of carbonworking electrodes, it is to be appreciated that other types ofelectrodes may be similarly enhanced according to the disclosure herein.Electrode types that may be enhanced through use of the disclosureherein also include gold, platinum, PEDOT, and the like.

Before describing the analyte sensors of the present disclosure andtheir enhancements in further detail, a brief overview of suitable invivo analyte sensor configurations and sensor systems employing theanalyte sensors will be provided first so that the embodiments of thepresent disclosure may be better understood. FIG. 1 shows a diagram ofan illustrative sensing system that may incorporate an analyte sensor ofthe present disclosure. As shown, sensing system 100 includes sensorcontrol device 102 and reader device 120 that are configured tocommunicate with one another over local communication path or link 140,which may be wired or wireless, uni- or bi-directional, and encrypted ornon-encrypted. Reader device 120 may constitute an output medium forviewing analyte concentrations and alerts or notifications determined bysensor 104 or a processor associated therewith, as well as allowing forone or more user inputs, according to some embodiments. Reader device120 may be a multi-purpose smartphone or a dedicated electronic readerinstrument. While only one reader device 120 is shown, multiple readerdevices 120 may be present in certain instances.

Reader device 120 may also be in communication with remote terminal 170and/or trusted computer system 180 via communication path(s)/link(s) 141and/or 142, respectively, which also may be wired or wireless, uni- orbi-directional, and encrypted or non-encrypted. Reader device 120 mayalso or alternately be in communication with network 150 (e.g., a mobiletelephone network, the internet, or a cloud server) via communicationpath/link 151. Network 150 may be further communicatively coupled toremote terminal 170 via communication path/link 152 and/or trustedcomputer system 180 via communication path/link 153. Alternately, sensor104 may communicate directly with remote terminal 170 and/or trustedcomputer system 180 without an intervening reader device 120 beingpresent. For example, sensor 104 may communicate with remote terminal170 and/or trusted computer system 180 through a direct communicationlink to network 150, according to some embodiments, as described in U.S.Patent Application Publication 2011/0213225 and incorporated herein byreference in its entirety.

Any suitable electronic communication protocol may be used for each ofthe communication paths or links, such as near field communication(NFC), radio frequency identification (RFID), BLUETOOTH® or BLUETOOTH®Low Energy protocols, WiFi, or the like. Remote terminal 170 and/ortrusted computer system 180 may be accessible, according to someembodiments, by individuals other than a primary user who have aninterest in the user's analyte levels. Reader device 120 may comprisedisplay 122 and optional input component 121. Display 122 may comprise atouch-screen interface, according to some embodiments.

Sensor control device 102 includes sensor housing 103, which may housecircuitry and a power source for operating sensor 104. Optionally, thepower source and/or active circuitry may be omitted. A processor (notshown) may be communicatively coupled to sensor 104, with the processorbeing physically located within sensor housing 103 or reader device 120.Sensor 104 protrudes from the underside of sensor housing 103 andextends through adhesive layer 105, which is adapted for adhering sensorhousing 103 to a tissue surface, such as skin, according to someembodiments.

Sensor 104 is adapted to be at least partially inserted into a tissue ofinterest, such as within the dermal or subcutaneous layer of the skin.Alternately, sensor 104 may be adapted to penetrate the epidermis. Stillfurther alternately, sensor 104 may be disposed superficially and notpenetrate a tissue, such as when assaying one or more analytes inperspiration upon the skin. Sensor 104 may comprise a sensor tail ofsufficient length for insertion to a desired depth in a given tissue.The sensor tail may comprise at least one working electrode and anactive area comprising an enzyme or enzyme system configured forassaying one or more analytes of interest.

A counter electrode may be present in combination with the at least oneworking electrode, optionally in further combination with a referenceelectrode. Particular electrode configurations upon the sensor tail aredescribed in more detail below in reference to FIGS. 2A-4. One or moreenzymes in the active area may be covalently bonded to a polymercomprising the active area, according to various embodiments.Alternately, enzymes may be non-covalently associated within the activearea, such as through encapsulation or physical entrainment. The one ormore analytes may be monitored in any biological fluid of interest suchas dermal fluid, interstitial fluid, plasma, blood, lymph, synovialfluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amnioticfluid, or the like. In particular embodiments, analyte sensors of thepresent disclosure may be adapted for assaying dermal fluid orinterstitial fluid to determine analyte concentrations in vivo. It is tobe appreciated, however, that the entirety of sensor control device 102may have one or more various configurations permitting fulltransplantation beneath tissue and into one or more body fluids forassaying one or more analytes of interest, without departing from thescope of the present disclosure.

Referring again to FIG. 1, sensor 104 may automatically forward data toreader device 120. For example, analyte concentration data may becommunicated automatically and periodically, such as at a certainfrequency as data is obtained or after a certain time period has passed,with the data being stored in a memory until transmittal (e.g., everyminute, five minutes, or other predetermined time period), such as byBLUETOOTH® or BLUETOOTH® Low Energy protocols. Data associated withdifferent analytes may be forwarded at the same frequency or differentfrequencies and/or using the same or different communication protocols.In other embodiments, sensor 104 may communicate with reader device 120in a non-automatic manner and not according to a set schedule. Forexample, data may be communicated from sensor 104 using RFID technologywhen the sensor electronics are brought into communication range ofreader device 120. Until communicated to reader device 120, data mayremain stored in a memory of sensor 104. Thus, a user does not have tomaintain close proximity to reader device 120 at all times, and caninstead upload data at a convenient time, automatically ornon-automatically. In yet other embodiments, a combination of automaticand non-automatic data transfer may be implemented. For example, datatransfer may continue on an automatic basis until reader device 120 isno longer in communication range of sensor 104.

An introducer may be present transiently to promote introduction ofsensor 104 into a tissue. In illustrative embodiments, the introducermay comprise a needle or similar sharp, or a combination thereof. It isto be recognized that other types of introducers, such as sheaths orblades, may be present in alternative embodiments. More specifically,the needle or other introducer may transiently reside in proximity tosensor 104 prior to tissue insertion and then be withdrawn afterward.While present, the needle or other introducer may facilitate insertionof sensor 104 into a tissue by opening an access pathway for sensor 104to follow. For example, the needle may facilitate penetration of theepidermis as an access pathway to the dermis to allow implantation ofsensor 104 to take place, according to one or more embodiments. Afteropening the access pathway, the needle or other introducer may bewithdrawn so that it does not represent a sharps hazard. In illustrativeembodiments, suitable needles may be solid or hollow, beveled ornon-beveled, and/or circular or non-circular in cross-section. In moreparticular embodiments, suitable needles may be comparable incross-sectional diameter and/or tip design to an acupuncture needle,which may have a cross-sectional diameter of about 250 microns. It is tobe recognized, however, that suitable needles may have a larger orsmaller cross-sectional diameter if needed for particular applications.For example, needles having a cross-sectional diameter ranging fromabout 300 microns to about 400 microns may be used.

In some embodiments, a tip of the needle (while present) may be angledover the terminus of sensor 104, such that the needle penetrates atissue first and opens an access pathway for sensor 104. In otherillustrative embodiments, sensor 104 may reside within a lumen or grooveof the needle, with the needle similarly opening an access pathway forsensor 104. In either case, the needle may be subsequently withdrawnafter facilitating sensor insertion.

Sensor configurations featuring a single active area that is configuredfor detection of a corresponding single analyte may employ two-electrodeor three-electrode detection motifs, as described further herein inreference to FIGS. 2A-2C. Sensor configurations featuring two differentactive areas for detection of separate analytes, either upon separateworking electrodes or upon the same working electrode, are describedseparately thereafter in reference to FIGS. 3A-4. Sensor configurationshaving multiple working electrodes may be particularly advantageous forincorporating two different active areas within the same sensor tail,since the signal contribution from each active area may be determinedmore readily through separate interrogation of each working electrode.Each active area may be overcoated with a mass transport limitingmembrane of the same or different composition.

When a single working electrode is present in an analyte sensor,three-electrode sensor configurations may comprise a working electrode,a counter electrode, and a reference electrode. Related two-electrodesensor configurations may comprise a working electrode and a secondelectrode, in which the second electrode may function as both a counterelectrode and a reference electrode (i.e., a counter/referenceelectrode). The various electrodes may be at least partially stacked(layered) upon one another and/or laterally spaced apart from oneanother upon the sensor tail. In any of the sensor configurationsdisclosed herein, the various electrodes may be electrically isolatedfrom one another by a dielectric material or similar insulator.

Analyte sensors featuring multiple working electrodes may similarlycomprise at least one additional electrode. When one additionalelectrode is present, the one additional electrode may function as acounter/reference electrode for each of the multiple working electrodes.When two additional electrodes are present, one of the additionalelectrodes may function as a counter electrode for each of the multipleworking electrodes and the other of the additional electrodes mayfunction as a reference electrode for each of the multiple workingelectrodes.

Any of the working electrode configurations described hereinafter maybenefit from the further disclosure below directed to decreasing theavailability of edge asperities of the working electrode upon the sensortail.

FIG. 2A shows a diagram of an illustrative two-electrode analyte sensorconfiguration, which is compatible for use in the disclosure herein. Asshown, analyte sensor 200 comprises substrate 212 disposed betweenworking electrode 214 and counter/reference electrode 216. Alternately,working electrode 214 and counter/reference electrode 216 may be locatedupon the same side of substrate 212 with a dielectric materialinterposed in between (configuration not shown). Active area 218 isdisposed as at least one layer upon at least a portion of workingelectrode 214. Active area 218 may comprise multiple discontiguous spotsor a single contiguous spot configured for detection of an analyte, asdiscussed further herein.

Referring still to FIG. 2A, membrane 220 overcoats at least active area218 and may optionally overcoat some or all of working electrode 214and/or counter/reference electrode 216, or the entirety of analytesensor 200, according to some embodiments. One or both faces of analytesensor 200 may be overcoated with membrane 220. Membrane 220 maycomprise one or more polymeric membrane materials having capabilities oflimiting analyte flux to active area 218 (i.e., membrane 220 is a masstransport limiting membrane having some permeability for the analyte ofinterest). The composition and thickness of membrane 220 may vary topromote a desired analyte flux to active area 218, thereby providing adesired signal intensity and stability. Analyte sensor 200 may beoperable for assaying an analyte by any of coulometric, amperometric,voltammetric, or potentiometric electrochemical detection techniques.

FIGS. 2B and 2C show diagrams of illustrative three-electrode analytesensor configurations, which are also compatible for use in thedisclosure herein. Three-electrode analyte sensor configurations may besimilar to that shown for analyte sensor 200 in FIG. 2A, except for theinclusion of additional electrode 217 in analyte sensors 201 and 202(FIGS. 2B and 2C). With additional electrode 217, counter/referenceelectrode 216 may then function as either a counter electrode or areference electrode, and additional electrode 217 fulfills the otherelectrode function not otherwise accounted for. Working electrode 214continues to fulfill its original function. Additional electrode 217 maybe disposed upon either working electrode 214 or electrode 216, with aseparating layer of dielectric material in between. For example, asdepicted in FIG. 2B, dielectric layers 219 a, 219 b, and 219 c separateelectrodes 214, 216, and 217 from one another and provide electricalisolation. Alternately, at least one of electrodes 214, 216, and 217 maybe located upon opposite faces of substrate 212, as shown in FIG. 2C.Thus, in some embodiments, electrode 214 (working electrode) andelectrode 216 (counter electrode) may be located upon opposite faces ofsubstrate 212, with electrode 217 (reference electrode) being locatedupon one of electrodes 214 or 216 and spaced apart therefrom with adielectric material. Reference material layer 230 (e.g., Ag/AgCl) may bepresent upon electrode 217, with the location of reference materiallayer 230 not being limited to that depicted in FIGS. 2B and 2C. As withsensor 200 shown in FIG. 2A, active area 218 in analyte sensors 201 and202 may comprise multiple spots or a single spot. Additionally, analytesensors 201 and 202 may likewise be operable for assaying an analyte byany of coulometric, amperometric, voltammetric, or potentiometricelectrochemical detection techniques.

Like analyte sensor 200, membrane 220 may also overcoat active area 218,as well as other sensor components, in analyte sensors 201 and 202,thereby serving as a mass transport limiting membrane. Additionalelectrode 217 may be overcoated with membrane 220 in some embodiments.Membrane 220 may again be produced through dip coating or in situphotopolymerization and vary compositionally or be the samecompositionally at different locations. Although FIGS. 2B and 2C havedepicted all of electrodes 214, 216, and 217 as being overcoated withmembrane 220, it is to be recognized that only working electrode 214 oractive area 218 may be overcoated in some embodiments. Moreover, thethickness of membrane 220 at each of electrodes 214, 216, and 217 may bethe same or different. As in two-electrode analyte sensor configurations(FIG. 2A), one or both faces of analyte sensors 201 and 202 may beovercoated with membrane 220 in the sensor configurations of FIGS. 2Band 2C, or the entirety of analyte sensors 201 and 202 may beovercoated. Accordingly, the three-electrode sensor configurations shownin FIGS. 2B and 2C should be understood as being non-limiting of theembodiments disclosed herein, with alternative electrode and/or layerconfigurations remaining within the scope of the present disclosure.

FIG. 3A shows an illustrative configuration for sensor 203 having asingle working electrode with two different active areas disposedthereon. FIG. 3A is similar to FIG. 2A, except for the presence of twoactive areas upon working electrode 214: first active area 218 a andsecond active area 218 b, which are responsive to different analytes andare laterally spaced apart from one another upon the surface of workingelectrode 214. Active areas 218 a and 218 b may comprise multiple spotsor a single spot configured for detection of each analyte. Thecomposition of membrane 220 may vary or be compositionally the same atactive areas 218 a and 218 b. First active area 218 a and second activearea 218 b may be configured to detect their corresponding analytes atworking electrode potentials that differ from one another, as discussedfurther below.

FIGS. 3B and 3C show cross-sectional diagrams of illustrativethree-electrode sensor configurations for sensors 204 and 205,respectively, each featuring a single working electrode having firstactive area 218 a and second active area 218 b disposed thereon. FIGS.3B and 3C are otherwise similar to FIGS. 2B and 2C and may be betterunderstood by reference thereto. As with FIG. 3A, the composition ofmembrane 220 may vary or be compositionally the same at active areas 218a and 218 b.

FIG. 4 shows a cross-sectional diagram of an illustrative analyte sensorconfiguration having two working electrodes, a reference electrode, anda counter electrode, which is compatible for use in the disclosureherein. As shown, analyte sensor 400 includes working electrodes 404 and406 disposed upon opposite faces of substrate 402. First active area 410a is disposed upon the surface of working electrode 404, and secondactive area 410 b is disposed upon the surface of working electrode 406.Counter electrode 420 is electrically isolated from working electrode404 by dielectric layer 422, and reference electrode 431 is electricallyisolated from working electrode 406 by dielectric layer 423. Outerdielectric layers 430 and 432 are positioned upon reference electrode431 and counter electrode 420, respectively. Membrane 440 may overcoatat least active areas 410 a and 410 b, according to various embodiments,with other components of analyte sensor 400 or the entirety of analytesensor 400 optionally being overcoated with membrane 440 as well. Again,membrane 440 may vary compositionally at active areas 410 a and 410 b,if needed, in order to afford suitable permeability values fordifferentially regulating the analyte flux at each location.

Alternative sensor configurations having multiple working electrodes anddiffering from the configuration shown in FIG. 4 may feature acounter/reference electrode instead of separate counter and referenceelectrodes 420, 431, and/or feature layer and/or membrane arrangementsvarying from those expressly depicted. For example, the positioning ofcounter electrode 420 and reference electrode 431 may be reversed fromthat depicted in FIG. 4. In addition, working electrodes 404 and 406need not necessarily reside upon opposing faces of substrate 402 in themanner shown in FIG. 4.

A carbon working electrode may suitably comprise the workingelectrode(s) in any of the analyte sensors disclosed herein. Whilecarbon working electrodes are very commonly employed in electrochemicaldetection, use thereof in electrochemical sensing is not withoutdifficulties. In particular, current related to an analyte of interestonly results when an active area interacts with an analyte and transferselectrons to the portion of the carbon working electrode adjacent to theactive area. Bodily fluid containing an analyte of interest alsointeracts with a carbon surface of the carbon working electrode notovercoated with an active area and does not contribute to the analytesignal, since there is no enzyme or enzyme system present at theselocations to facilitate electron transfer from the analyte to theworking electrode. Interferents may, however, undergo oxidation atportions of the working electrode lacking an active area and contributebackground to the overall signal. Thus, carbon working electrodes withan extraneous (or “exposed”) carbon area upon the electrode surface donot meaningfully contribute to the analyte signal and may lead tocontributory background signals in some cases. Other electrodes havingan excessive surface area not directly detecting an analyte of interestmay experience similar background signals and may be enhanced throughmodification of the disclosure herein.

Although various interferents may interact with the working electrode ofthe analyte sensors described herein, ascorbic acid is one example of aninterferent commonly present in biological fluids that may generate abackground signal at a carbon working electrode. For example, ascorbicacid oxidizes at the working electrode to produce dehydroascorbic acid.Various embodiments of the present disclosure will be described hereinwith reference to the interferent being ascorbic acid; however, it is tobe understood that that the embodiments and analyte sensorconfigurations described herein are equally applicable to otherinterferents (electroactive species within a bodily fluid having ananalyte of interest).

As provided above, the active area described herein may be a singlesensing layer, a sensing layer having multiple sensing spots, or asensing layer having multiple sensing spots compressed together and thusrepresenting essentially a single sensing layer. Referring now to FIG.5, illustrated is a top view of conventional carbon working electrode500 having an active area 504 disposed thereon comprising multiplesensing spots 518. Only portions of carbon working electrode 500comprising the sensing spots 518 contribute signal associated with ananalyte of interest when the analyte interacts with the active area 504.Although carbon working electrode 500 shows six sensing spots 518 withinthe active area 504, it is to be appreciated that fewer or greater thansix sensing spots 518 may be included upon carbon working electrode 500,without departing from the scope of the present disclosure. Extraneouscarbon area 510 is not directly overlaid with sensing spots 518 and doesnot contribute signal associated with the analyte but may generate abackground signal associated with one or more interferents. Accordingly,the oxidation of interferents at carbon working electrode 500 isproportional to the area of extraneous carbon area 510 available forinteraction with the interferents. Indeed, the oxidation of ascorbicacid at carbon working electrode 500 scales roughly linearly with thearea of available extraneous carbon area 510.

As shown, the active area 504 is discontiguous and in the form ofmultiple sensing spots 518. As defined herein, the term “discontiguous,”and grammatical variants thereof, means that any single spot (sensingelement) does not share an edge or boundary (e.g., is not touching) anadjacent spot.

The sensor tails described in the present disclosure comprising thecarbon working electrode 500 may be prepared upon a template substratematerial (see FIGS. 2A-2B, 3A-3C, 4) along with additional layeredelements of the sensor tail (e.g., dielectric materials, otherelectrodes, and the like). During sensor fabrication, the sensor tailcomprising the carbon working electrode 500 is thereafter singulated byone or more means. Singulation may be achieved by one or more cutting orseparation protocols including, but not limited to, laser singulation,slitting, shearing, punching, and the like. Singulation of the sensortails may be performed before or after application of the active areaupon the carbon working electrode 500 toward the distal tip of thesensor tail (i.e., the portion of the sensor tail that will be inserteddeepest into a tissue). As used herein, the distal “tip” of the sensortail is referred to as the most distal edge of a sensor tail, or thatportion that is most deeply inserted into a tissue.

One or more portions of the sensor tail are laser singulated, typicallyrequiring multiple laser passes, to cut the sensor tail into the desiredshape. The tip of the sensor tail comprises at least a portion of theworking electrode and the active area. Typically, the laser singulatedsensor tails have a width in the range of about 50 μm to about 800 μmand a length of about 1 mm to about 20 mm, such as a width in the rangeof about 100 μm to about 500 μm and a length of about 3 mm to about 10mm, encompassing any value and subset therebetween and in which theupper and lower limits are separable. Generally, the distal portion ofthe sensor tail accounts for a distal length of about 0.1 mm to about 10mm, such as about 0.1 mm to about 5 mm, encompassing any value andsubset therebetween and in which the upper and lower limits areseparable. After laser singulation, a mass transport limiting membraneis deposited upon at least the sensor tip comprising the active area.

In one or more aspects of the present disclosure, prior to disposing themass transport limiting membrane, carbon asperities may be present alongthe edges of the carbon electrode due to the laser singulation process.These carbon asperities may provide a surface upon which interferentsmay react and contribute background signal to an analyte sensor.

Laser singulation of a carbon working electrode may result in theformation of carbon asperities having widths of about 75 μm or less,such as in the range of about 1 μm to about 75 μm, or about 5 μm toabout 50 μm, or about 10 μm to about 30 μm, encompassing any value andsubset therebetween and in which the upper and lower limits areseparable. Further, these carbon asperities may have a height of about50 μm or less, or about 20 μm or less, such as in the range of about 1μm to about 50 μm, or about 1 μm to about 30 μm, or about 1 μm to about20 μm, or about 2 μm to about 10 μm, as described hereinbelow in greaterdetail, encompassing any value and subset therebetween and in which theupper and lower limits are separable. Accordingly, these carbonasperities may provide substantial area with which interferents mayinteract. In addition, the asperities can contribute to inconsistentcoverage (thickness) of a mass transport limiting membrane. These carbonasperities may be reduced or removed by one or more laser planingmethods, as described hereinbelow.

Referring first to FIG. 6A, and prior to any laser planing to reduce orremove carbon asperities in accordance with the present disclosure,illustrated is a close up of an example of a laser singulated carbonworking electrode for use as at least a portion of a sensor tail, inwhich the carbon working electrode has no mass transport limitedmembrane deposited thereon. Electrodes cut into their desired shape byother means may have asperities of a similar appearance and size. Carbonasperities are apparent along the edges of the working electrode withwhich interferents may react. FIG. 6B shows a depth profile along theline indicated in FIG. 6A, evaluated along the identified 430.71 μmprofile width. The 3D optical profile was obtained using a ZEGAGE™ 3DOptical Profiler, ZYGOO® Corporation (Middlefield, Conn.). As shown inFIG. 6B, carbon asperities along the singulation (ablation) edges of theexample singulated sensor tail are up to about 30 μm wide and up toabout 10 μm in height.

A mass transport limiting membrane may reduce or prevent interferentaccess to extraneous carbon areas (e.g., extraneous carbon area 510 ofFIG. 5). When disposed upon a laser singulated carbon working electrode(and an active area thereupon), the thickness of the membrane variesacross the width of the working electrode, particularly wheresignificant asperities are present. Typically, the membrane is thinnestalong the edges of the electrode, which is also where the carbonasperities are located. Accordingly, even when a membrane is present,the carbon asperities may not be sufficiently coated with the membraneto adequately reduce or prevent interferent interaction therewith.

Referring to FIG. 7A, and prior to any laser planing to reduce or removecarbon asperities in accordance with one or more aspects of the presentdisclosure, illustrated is a close up of an example laser singulatedcarbon working electrode having a mass transport limited membranedeposited thereon. FIG. 7B shows a depth profile along the lineindicated in FIG. 7A, evaluated along the identified 345.53 μm profilewidth. The 3D optical profile was obtained using a ZEGAGE™ 3D OpticalProfiler, ZYGOO® Corporation (Middlefield, Conn.). As shown in FIG. 7B,the membrane is considerably thinner along the singulation ridges of thecarbon working electrode.

In various aspects, the present disclosure provides methods and analytesensors in which carbon working electrodes for use in forming a sensortail are planed by one or more single- or multi-pass laser planing cuts,alone or in combination with the additional enhancements describedherein. In some embodiments, a single-pass laser planing method is usedin which the laser depth is set to less than the thickness of theworking electrode. For example, the laser planing may remove the topportions of the carbon layer, such as the top about 50% of the carbonlayer. The carbon layer is typically in the range of 5 μm to about 20 μm(without asperities); in some embodiments, about 5 μm to about 10 μm(e.g., about 20%, 30%, 40%, 50%, 60%, or 70%, up to 100%) may be removedtherefrom (e.g., see FIG. 13C). Laser planing according to thedisclosure herein may remove or decrease the prominence of asperities.

In some embodiments, greater than 1, such as less than about 20 (orabout 15, or about 10), single-pass laser planing cuts may be made, eachprogressively closer to the midline length of the working electrode toreduce or eliminate the carbon asperities. In such a way, initial laserplaning cuts may be made at the outermost location of any single carbonasperity and subsequent laser planing cuts may be made toward themidline length of the working electrode to create a milled edge, whichmay be a stepped edge of approximately 90° or beveled edge (i.e., anedge that is not perpendicular to the faces of the electrode) if, forexample, the most proximal laser planing cut toward the midline of theelectrode does not result in a true 90° angle (see FIG. 8, laser planingcut (edge) 810 shown as a sloped edge rather than a shear 90° angleedge). For example, in one embodiment, about 1 to about 20, such asabout 2 to about 10, single-pass laser planing cuts may be made, eachhaving a distance apart between about 0.1 μm to about 200 such as about1 μm to about 100 encompassing any value and subset therebetween and inwhich the upper and lower limits are separable. Selection of theparticular number of laser planing passes and their distance apart maybe based on a number of factors including, but not limited to, the shapeand size of the carbon asperities, the length and width of the workingelectrode, the coverage profile of any membrane disposed thereupon, andthe like, and any combination thereof.

Laser planing may be preferentially used to remove at least about 5% upto about 100% of the total carbon asperity area from a singulated sensortail comprising a carbon working electrode, encompassing any value andsubset therebetween and in which the upper and lower limits areseparable. In some embodiments, up to 100% of the carbon asperities areremoved, or about 5% to about 75%, or about 5% to about 50%, or about 5%to about 25% of the total carbon asperity area are removed, encompassingany value and subset therebetween and in which the upper and lowerlimits are separable. In preferred embodiments, at least about 50% ofthe carbon asperities total area are removed. The particular amount ofcarbon asperity removal may be based on a number of factors including,but not limited to, the density, shape, and size of the carbonasperities, the concentration of analyte of interest compared to theconcentration of interferent available within the bodily fluid beingassayed and the like, and any combination thereof.

FIG. 8 shows a photograph of an edge of a sensor tail 800 showing lasersingulation cut (ridge) 805 and laser planing cut (edge) 810 recessedfrom the edge of the sensor tail to remove a portion of the edge of acarbon working electrode (the carbon or electrode layer), in accordancewith one or more embodiments of the present disclosure. That is, thelaser planing cut 810 is directed to reducing the carbon asperitiesalong the upper or top portion of the carbon electrode (where the activearea resides, for example), while a thinner portion of the workingelectrode remains along an outer perimeter (and at the opposite portionof the electrode, which does not comprise the active area).

In one or more aspects of the present disclosure, alone or incombination with any other enhancements to reduce or eliminate analytesensor signals associated with interferents, provided is an analytesensor comprising an interferent-reactant species. As used herein, theterm “interferent-reactant species,” and grammatical variants thereof,refers to any compound, whether biological or non-biological, that arecapable of reacting with an interferent and rendering it inactive suchthat it cannot contribute to the measured signal at the workingelectrode. That is, the interferent-reactant species may be included aspart of an analyte sensor in order to “pre-react” an interferent beforeit is able to react on (contact) the working electrode of the analytesensor. Accordingly, the interferent-reactant species can eliminate orreduce the local concentration of an interferent present at oraccessible to the working electrode, thereby eliminating or reducingsignal attributed to such interferents because the interferents neverreach excess area of a working electrode.

Various aspects of the methods and analyte sensors integrating aninterferent-reactant species are described with reference tointerferent-reactant species for ascorbic acid elimination or removal;it is to be continually appreciated that the enhancements describedherein pertain to other potential interferents, without limitation. Suchinterferents may include, for example, ascorbic acid (vitamin C),glutathione, uric acid, paracetamol (acetaminophen), isoniazid,salicylate, and the like, and any combination thereof. In non-limitingexamples, the interferent-reactant species of the present disclosure maybe an enzyme of ascorbate oxidase (to react with ascorbic acid),glutathione peroxidase (to react with glutathione), xanthine oxidase (toreact with uric acid), urate oxidase (to react with uric acid),cytochrome P450 (to react with paracetamol), eosinophil peroxidase (toreact with isoniazid), salicylate-oxidizing enzyme (to react withsalicylate), other enzymes that can oxidize, reduce, or otherwise reactand decompose the interferent of interest, and the like, and anycombination thereof. In alternative or combination embodiments, theinterferent-reactant species may be one of a non-enzyme. For example,various metal oxides, such as manganese oxide (MnO₂) or iron oxide(Fe₂CO₃) may oxidize or otherwise react and decompose ascorbic acid andbe used as the one or more interferent-reactant species of the presentdisclosure.

Referring to FIG. 9A, illustrated is a depiction of a conventionalsensor 900 demonstrating potential interferent reaction of ascorbic acid902 with excess working electrode and, potentially, also the sensingchemistry, thereby producing signal attributable to the ascorbic acid.The sensor of FIG. 9A has no interferent-reactant species incorporatedtherewith. The ascorbic acid 902 encounters the sensor 900 throughbodily fluid 904 (e.g., interstitial fluid) and contacts sensingchemistry 906 and the excess area working electrode 908 (e.g., thatwhich does not include sensing chemistry 906) disposed upon substrate910. Upon encountering the sensing chemistry 906 and working electrode908, the ascorbic acid 902 may be oxidized at least on the excessworking electrode 908 and/or additionally on the sensing chemistry 908and converted to dehydroascorbic acid 912.

According to various aspects of the present disclosure, FIG. 9Billustrates a depiction of the sensor of FIG. 9A incorporating aninterferent-reactant species 914, and in particular theinterferent-reactant species of ascorbate oxidase (AOx). As shown, theascorbate oxidase 914 reacts with the ascorbic acid 902 prior to itcontacting the working electrode 906 or sensing chemistry 906, therebypreventing said ascorbic acid 902 from contributing to analyte signal.It is to be noted that the sensor depicted in FIG. 9B may have anyconfiguration and/or component of the sensors described herein, withoutlimitation.

The particular location of one or more interferent-reactant species forincorporation into the analyte sensors of the present disclosure is notconsidered to be particularly limiting. For example, theinterferent-reactant species may be provided as part of an analytesensing active area; a membrane coating an analyte sensing active area;as its own layer atop any of a working electrode, analyte sensing activearea, and/or membrane coating; and the like; and any combinationthereof. When provided as part of an active layer, membrane, or its ownlayer, it may be free-floating or otherwise immobilized (e.g.,covalently or non-covalently bound) within a polymer matrix. Theparticular concentration of the interferent-reactant speciesincorporated into an analyte sensor (in any one or more locations) maydepend on a number of factors including, but not limited to, theparticular analyte(s) of interest, the particular interferent(s) ofinterest, the in vivo location of the analyte sensor, and the like, andany combination thereof. In some instances, when theinterferent-reactant species is an enzyme, the total amount ofinterferent-reactant species may be in the range of about 0.01 Units toabout 200 Units of activity per sensor, such as about 0.01 Units toabout 100 Units of activity per sensor, encompassing any value andsubset therebetween and in which the upper and lower limits areseparable. For example, a sensor having an interferent-reactant speciesof ascorbate oxidase may have about 0.01 Units to about 10 Units, orabout 0.01 Units to about 5 Units, or about 0.1 Units to about 1 Unitsof activity per sensor. In other instances, when theinterferent-reactant species is a non-enzyme compound, such as a metaloxide, the total amount of interferent-reactant species may be in therange of about 0.01 μg to about 200 μg per sensor, or about 0.1 μg toabout 100 μg per sensor, encompassing any value and subset therebetweenand in which the upper and lower limits are separable. For example, asensor having an interferent-reactant species of MnO₂ may be present inan amount of about 0.1 μg to about 10 μg per sensor and in which theupper and lower limits are separable.

As stated above, generally, the interferent-reactant species describedherein, whether present as a layer itself, present within the membrane,or present within an active area will be present within a polymermatrix, either mobilized or immobilized. This polymer matrix may becomposed of any polymers, crosslinkers, and/or additives compatible withthe interferent-reactant species selected for use in the analyte sensorthat does not interfere with the sensing chemistry. Each of thepolymers, crosslinkers, and/or additives may be selected from any ofthose described herein, without limitation. For example, non-limitingexamples of such polymers include poly(4-vinylpyridine) andpoly(N-vinylimidazole) (PVI) or a copolymer thereof, a sulfonatedtetrafluoroethylene based fluoropolymer-copolymer (e.g., NAFION™, TheChemours Company, Wilmington, Del.), polyvinyl alcohol, and anycombination thereof; non-limiting examples of crosslinkers includetriglycidyl glycerol ether (gly3) and/or PEDGE and/orpolydimethylsiloxane diglycidylether (PDMS-DGE); non-limiting examplesof additives include stabilizers, such as albumin, and/or any otherstabilizers described herein.

In one or more aspects of the present disclosure, alone or incombination with any other enhancements to reduce or eliminate analytesensor signals associated with interferents, provided is an analytesensor comprising a scrubbing electrode (with or without integration ofan interferent-reactant species and/or asperity planing, for example).As described herein, the term “scrubbing electrode,” and grammaticalvariants thereof, refers to an electrode capable of reacting with aninterferent to render it inactive such that it cannot contribute to themeasured signal at the working electrode. That is, the scrubbingelectrode may be included as part of an analyte sensor in order to“pre-react” an interferent before it is able to react on the workingelectrode of the analyte sensor. Accordingly, similar to the presence ofan interferent-reactant species, the scrubbing electrode can eliminateor reduce the local concentration of an interferent present at oraccessible to the working electrode, thereby eliminating or reducingsignal attributed to such interferents because the interferents neverreach excess area of a working electrode.

In one or more aspects, the scrubbing electrode may be positioned in afacing relationship, and spatially offset from the working electrode.That is, the active area of the working electrode and the active area ofthe scrubbing electrode, which may or may not be disposed on asubstrate, face one another and are separated by a gap. Preferably, thegap is a thin layer between the two electrodes that permits bodilyfluids to pass therebetween, including the analyte of interest and anyinterferent(s) therein. The configuration of the scrubbing electroderelative to the working electrode is desirably such that the bodilyfluid comes into contact with the scrubbing electrode for a sufficienttime to react to any interferent prior to the bodily fluid reaching theworking electrode. The scrubbing electrode does not comprise any sensingchemistry and, accordingly, analytes of interest do not react therewith.In such a manner, the bodily fluid has been rid or substantially rid ofthe interferent, and the signal obtained at the working electrode isattributable entirely or primarily to the analyte of interest.

Various electrode configurations may be used to ensure that bodily fluidcontacts the scrubbing electrode prior to the working electrode. Onesuch non-limiting configuration 1000 is shown in FIG. 10. As shown, ascrubbing electrode 1006 and a working electrode 1008 are in facingrelationship and the working electrode 1008 is recessed, or otherwise ofa lesser width, compared to the scrubbing electrode 1006. The workingelectrode further comprises sensing chemistry disposed thereon (notshown). Although the particular configuration of the working electrode1008 and scrubbing electrode 1006 shown in FIG. 10 is in the shape of arectangle, other configurations may be equally applicable to theembodiments described herein, such as square, round, helical, and thelike. Generally, the working electrode 1008 and the scrubbing electrode1006 may have a length 1004 that is greater than its width 1002.

In one or more aspects, the width of the scrubbing electrode to theworking electrode may be in the range of about 2:1 to about 50:1,encompassing any value and subset therebetween and in which the upperand lower limits are separable. For example, in some instances, thescrubbing electrode 1006 may have a width in the range of about 200 μmto about 8000 such as about 300 μm to about 5000 and the workingelectrode 1008 may have a width in the range of about 50 μm to about2000 such as about 50 μm to about 1000 encompassing any value and subsettherebetween and in which the upper and lower limits are separable.These dimensions incorporate orientations in which a thin layer 1010 mayextend up the length of the sensor tail, having a linear or non-linearshape, in order to increase the ratio between the size of scrubbingelectrode 1006 and the size of the working electrode 1008, withoutmaking the sensor tail too wide for practical in vivo use (insertion).

The thin layer 1010 is formed between the scrubbing electrode 1006 andthe working electrode 1008. This thin layer may be in the range of about1 μm to about 200 such as about 10 μm to about 100 encompassing anyvalue and subset therebetween and in which the upper and lower limitsare separable. In some instances, the thin layer may be about 10 μm toabout 50 or about 10 about 20 about 30 about 40 or about 50 μm and inwhich the upper and lower limits are separable. The thin layer 1010 isgenerally formed by sealing fluid passage along two opposing edges ofthe scrubbing electrode 1006 (e.g., a thin layer “cell”), such thatbodily fluid can enter the space between the unsealed thin layer 1010space in a controlled fashion to ensure that it reaches the scrubbingelectrode 1006 prior to the working electrode 1008. In general, a largerratio between the scrubbing electrode 1006 surface area to the thinlayer 1010 volume may be preferred to maximize the opportunity forsolutes (e.g., interferents) to interact with the scrubbing electrode1006. For example, with reference to FIG. 10, the thin layer 1010between the scrubbing electrode 1006 and the working electrode 1008 maybe formed by applying an adhesive, spacer, or other non-limitingseparation means along the width edges of the electrodes. As such,bodily fluid is directed through the edges along the length and into thethin layer 1010. Accordingly, when bodily fluid, including interferentsand the analyte of interest, diffuse through the thin-layer 1010, thereis ample interaction with the scrubbing electrode 1006 before reachingthe working electrode 1008. As such, analyte sensors comprising suchscrubbing electrodes 1006 need not, although may, rely on a membrane tolimit interferent interaction with the working electrode 1008, which mayprovide manufacturing and cost benefits.

In various embodiments, the thin layer 1010 may be modified with asurfactant, hydrogel, membrane, or other material aid in channeling thebodily fluid into the thin layer 1010, to aid in biocompatibility, toprovide a microbicidal or microstatic quality, and the like, and anycombination thereof.

In one or more aspects, various configurations may be tuned such thatthe scrubbing electrode may be independently controlled, such as byadjusting the scrubbing electrode potential in order to fine-tune itsreaction effectiveness with particular interferents. In general, theeffectiveness of the scrubbing electrode to react with interferents willincrease with higher potentials. The scrubbing electrode potential maybe in the range of about −2000 mV to about +2000 mV, such as about −1000mV to about +1000 mV, encompassing any value and subset therebetween andin which the upper and lower limits are separable. In general, thescrubbing electrode potential may be any working potential within thepotential window of water; that is, the potential at which water, therelevant solvent for bodily fluids, is not itself oxidized or reduced.The scrubbing electrode potential may be relative to an includedreference electrode (e.g., a Ag/AgCl reference electrode), which may beshared by both the scrubbing electrode and working electrode, in someembodiments. Furthermore, running the scrubbing electrode at generallynegative potentials may enable the additional scrubbing of oxidizingagents, such as oxygen, which may be beneficial depending on the analyteof interest. That is, the scrubbing electrode may be used to scavengeoxygen to decrease its contribution to analyte signal.

The composition of the scrubbing electrode is not considered to beparticularly limiting and may be made of known electrode materials, andmay be the same or of different composition than the working electrode.Examples of suitable materials include, but are not limited to, carbon,gold, platinum, PEDOT, and the like. In some instances, the compositionof the scrubbing electrode may be modified or supplemented with amaterial specific for reaction with an interferent of interest or toincrease the surface area of the scrubbing electrode, among otheradvantages. It is further to be appreciated that an interferent-reactantspecies may be coated upon the scrubbing electrode in any manner, asdescribed hereinabove, in order to further enhance the elimination orreduction of interferents reaching the working electrode.

In some embodiments, rather than having a thin layer configuration forincorporation of a scrubbing electrode, the scrubbing electrodecomposition may be selected such that it is permeable to the analyte ofinterest. In such a manner, the scrubbing electrode may be layered abovethe working electrode, having an analyte permeable membrane ordielectric layer therebetween to avoid shorting of the sensor, and nothin layer. That is, an insulating material that is itself permeable tothe analyte of interest is disposed between the permeable scrubbingelectrode and the working electrode comprising the analyte sensingmaterial. In such a manner, and based on the same rationale as the thinlayer scrubbing electrode configurations described above, bodily fluid,comprising both the analyte of interest and interferents, will come intocontact with the permeable scrubbing electrode where interferents reactand are eliminated or otherwise reduced in concentration prior to thebodily fluid (comprising the analyte of interest and no or lessinterferents) coming into contact with the working electrode. Therefore,the scrubbing electrode can eliminate or reduce the local concentrationof an interferent present at or accessible to the working electrode,thereby eliminating or reducing signal attributed to such interferentsbecause the interferents never reach excess area of a working electrode.

One such non-limiting configuration of an analyte sensor 1100 comprisinga permeable scrubbing electrode is shown in FIG. 11. As shown, a workingelectrode 1102 comprises sensing chemistry disposed thereupon to form anactive area 1104 (as a single area or comprising multiple discontiguousspots). Upon the active area 1104 is an analyte-permeable insulatinglayer 1106, which may be of any material, such as any of the polymersdescribed herein, provided that one or more analytes of interest 1112can diffuse therethrough. For example the analyte-permeable insulatinglayer 1106 may be a diffusion-limiting membrane. An analyte-permeablescrubbing electrode 1108 is disposed upon the analyte-permeableinsulating layer 1106. While the analyte-permeable scrubbing electrode1108 may be the same dimensions as the base working electrode 1102, inpreferred embodiments, the analyte-permeable scrubbing electrode 1108has a shape and size that contacts bodily fluid prior to either of theinsulating layer 1106 or the working electrode 1102. An outer membrane1110 may be included to provide additional diffusion-limiting qualities,biocompatibility qualities, microbicidal or microstatic qualities,protection of the permeable scrubbing electrode 1108, and the like, andany combination thereof. As shown in FIG. 11, an interferent 1114 candiffuse through the outer membrane 1110 to the permeable scrubbingelectrode 1106, where it reacts and is rendered inactive such that itcannot contribute to the measured signal at the working electrode 1102.Differently, the analyte of interest 1112 is not reactive with thescrubbing electrode 1106 (which has no analyte sensing chemistry) andthe analyte 1112 diffuses through the outer membrane 1110, the scrubbingelectrode 1108, and the insulating layer 1106 to the sensing layer 1104upon the working electrode 1102. Another non-limiting configuration, asshown in FIG. 23 discussed below, may employ a “well” structure havingan analyte-permeable scrubbing electrode.

Another non-limiting configuration of an analyte sensor 1200 comprisinga permeable scrubbing electrode is shown in FIG. 12. In thisconfiguration, the permeable scrubbing electrode 1202 is provided incombination with a non-permeable scrubbing electrode 1204 trace toprovide electrical contact such that a potential can be applied to thepermeable scrubbing electrode 1202. The non-permeable scrubbingelectrode 1204 may be traced upon dielectric material 1206, disposedworking electrode 1208 and itself disposed a base substrate 1210 (e.g.,a plastic base substrate). A second dielectric may be further disposedupon non-permeable scrubbing electrode 1204. Sensing chemistry 1204 maybe dispensed upon an exposed portion of the working electrode 1208. Theportion of the sensor A may be produced and singulated. Thereafter, itmay be dip-coated to apply an inner polymer membrane 1214 and cured,then dip-coated to apply the permeable scrubbing membrane 1202 andcured, then finally dip-coated to apply an outer polymer membrane 1216.This configuration may provide manufacturing and cost benefits.

Each of the various compositions of the common layers and elements ofthe sensors described herein may be equally included in any or allembodiments comprising an analyte-permeable scrubbing electrode. Thecomposition of the analyte-permeable scrubbing electrode is notconsidered to be particularly limiting, provided that it is conductive,able to react with an interferent (e.g., oxidize ascorbic acid), andpermeable to the particular analyte of interest. In some instances, thepermeable electrode may be composed of a carbon nanotube material. Otherformulations may include, but are not limited to, conductivenanoparticles, conductive nanowires, and the like, and any combinationthereof. The permeable scrubbing electrode may further be supplementedwith other conductive inks or polymers to enhance conductivity, enhancepermeability, enhance the physical properties of the permeableelectrode, and the like, and any combination thereof. For example,poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) maybe incorporated or impregnated with a carbon nanotube permeablescrubbing electrode composition to increase its viscosity to enhancedip-coating. In one or more aspects, electron transfer agents, such asthose described herein, may be incorporated or otherwise impregnatedinto the porous structure of an analyte-permeable scrubbing electrode toenhance interferent scrubbing efficiency.

The thickness of an analyte-permeable scrubbing electrode is notconsidered to be particularly limiting and may be in the range of about0.5 μm to about 100 such as about 1 μm to about 50 encompassing anyvalue and subset therebetween and in which the upper and lower limitsare separable. Without being bound by theory, the thickness of thepermeable scrubbing electrode may be increased to enhance scrubbingefficiency as interferents would be exposed to a greater surface area ofthe scrubbing electrode, provided that the thickness does not adverselyinterfere with diffusion of the analyte of interest.

Without being bound by theory, in some embodiments, the scrubbingelectrode (whether or not permeable) may additionally be used toregenerate the product of the analyte detection system, therebyincreasing the concentration of analytes and effectively amplifying theanalyte signal.

The various layers of any of the aforementioned components of theanalyte sensors described herein may be deposited by any suitable means,such as, without limitation, automated dispensing or dip-coating.Electrodes may be screen printed, for example, and traces provided tomake appropriate electrical connections.

Active areas within any of the analyte sensors disclosed herein maycomprise one or more analyte-responsive enzymes, either acting alone orin concert within an enzyme system. One or more enzymes may becovalently bonded to a polymer comprising the active area, as can one ormore electron transfer agents located within the active area.

Examples of suitable polymers within each active area may includepoly(4-vinylpyridine) and poly(N-vinylimidazole) or a copolymer thereof,for example, in which quaternized pyridine and imidazole groups serve asa point of attachment for an electron transfer agent or enzyme(s). Othersuitable polymers that may be present in the active area include, butare not limited to, those described in U.S. Pat. No. 6,605,200,incorporated herein by reference in its entirety, such as poly(acrylicacid), styrene/maleic anhydride copolymer, methylvinylether/maleicanhydride copolymer (GANTREZ polymer), poly(vinylbenzylchloride),poly(allylamine), polylysine, poly(4-vinylpyridine) quaternized withcarboxypentyl groups, and poly(sodium 4-styrene sulfonate).

Enzymes covalently bound to the polymer in the active areas that arecapable of promoting analyte detection are not believed to beparticularly limited. Suitable enzymes may include those capable ofdetecting glucose, lactate, ketones, creatinine, or the like. Any ofthese analytes may be detected in combination with one another inanalyte sensors capable of detecting multiple analytes. Suitable enzymesand enzyme systems for detecting these analytes are describedhereinafter.

In some embodiments, the analyte sensors may comprise aglucose-responsive active area comprising a glucose-responsive enzymedisposed upon the sensor tail. Suitable glucose-responsive enzymes mayinclude, for example, glucose oxidase or a glucose dehydrogenase (e.g.,pyrroloquinoline quinone (PQQ) or a cofactor-dependent glucosedehydrogenase, such as flavine adenine dinucleotide (FAD)-dependentglucose dehydrogenase or nicotinamide adenine dinucleotide(NAD)-dependent glucose dehydrogenase). Glucose oxidase and glucosedehydrogenase are differentiated by their ability to utilize oxygen asan electron acceptor when oxidizing glucose; glucose oxidase may utilizeoxygen as an electron acceptor, whereas glucose dehydrogenases transferelectrons to natural or artificial electron acceptors, such as an enzymecofactor. Glucose oxidase or glucose dehydrogenase may be used topromote detection. Both glucose oxidase and glucose dehydrogenase may becovalently bonded to a polymer comprising the glucose-responsive activearea and exchange electrons with an electron transfer agent (e.g., anosmium (Os) complex or similar transition metal complex), which may alsobe covalently bonded to the polymer. Suitable electron transfer agentsare described in further detail below. Glucose oxidase may directlyexchange electrons with the electron transfer agent, whereas glucosedehydrogenase may utilize a cofactor to promote electron exchange withthe electron transfer agent. FAD cofactor may directly exchangeelectrons with the electron transfer agent. NAD cofactor, in contrast,may utilize diaphorase to facilitate electron transfer from the cofactorto the electron transfer agent. Further details concerningglucose-responsive active areas incorporating glucose oxidase or glucosedehydrogenase, as well as glucose detection therewith, may be found incommonly owned U.S. Pat. No. 8,268,143, for example, the entirety ofwhich is incorporated herein by reference.

In some embodiments, the active areas of the present disclosure may beconfigured for detecting ketones. Additional details concerning enzymesystems responsive to ketones may be found in commonly owned U.S. patentapplication Ser. No. 16/774,835 entitled “Analyte Sensors and SensingMethods Featuring Dual Detection of Glucose and Ketones,” filed on Jan.28, 2020, and published as U.S. Patent Application Publication2020/0237275, the entirety of which is incorporated herein by reference.In such systems, β-hydroxybutyrate serves as a surrogate for ketonesformed in vivo, which undergoes a reaction with an enzyme systemcomprising β-hydroxybutyrate dehydrogenase (HBDH) and diaphorase tofacilitate ketones detection within a ketones-responsive active areadisposed upon the surface of at least one working electrode, asdescribed further herein. Within the ketones-responsive active area,β-hydroxybutyrate dehydrogenase may convert β-hydroxybutyrate andoxidized nicotinamide adenine dinucleotide (NAD⁺) into acetoacetate andreduced nicotinamide adenine dinucleotide (NADH), respectively. It is tobe understood that the term “nicotinamide adenine dinucleotide (NAD)”includes a phosphate-bound form of the foregoing enzyme cofactors. Thatis, use of the term “NAD” herein refers to both NAD⁺ phosphate and NADHphosphate, specifically a diphosphate linking the two nucleotides, onecontaining an adenine nucleobase and the other containing a nicotinamidenucleobase. The NAD⁺/NADH enzyme cofactor aids in promoting theconcerted enzymatic reactions disclosed herein. Once formed, NADH mayundergo oxidation under diaphorase mediation, with the electronstransferred during this process providing the basis for ketonesdetection at the working electrode. Thus, there is a 1:1 molarcorrespondence between the amount of electrons transferred to theworking electrode and the amount of β-hydroxybutyrate converted.Transfer of the electrons to the working electrode may take place underfurther mediation of an electron transfer agent, such as an osmium (Os)compound or similar transition metal complex, as described in additionaldetail below. Albumin may further be present as a stabilizer within theactive area. The β-hydroxybutyrate dehydrogenase and the diaphorase maybe covalently bonded to a polymer comprising the ketones-responsiveactive area. The NAD⁺ may or may not be covalently bonded to thepolymer, but if the NAD⁺ is not covalently bonded, it may be physicallyretained within the ketones-responsive active area, such as with a masstransport limiting membrane overcoating the ketones-responsive activearea, wherein the mass transport limiting membrane is also permeable toketones.

Other suitable chemistries for enzymatically detecting ketones may beutilized in accordance with the embodiments of the present disclosure.For example, β-hydroxybutyrate dehydrogenase (HBDH) may again convertβ-hydroxybutyrate and NAD⁺ into acetoacetate and NADH, respectively.Instead of electron transfer to the working electrode being completed bydiaphorase and a suitable redox mediator, the reduced form of NADHoxidase (NADHOx (Red)) undergoes a reaction to form the correspondingoxidized form (NADHOx (Ox)). NADHOx (Red) may then reform through areaction with molecular oxygen to produce superoxide, which may undergosubsequent conversion to hydrogen peroxide under superoxide dismutase(SOD) mediation. The hydrogen peroxide may then undergo oxidation at theworking electrode to provide a signal that may be correlated to theamount of ketones that were initially present. The SOD may be covalentlybonded to a polymer in the ketones-responsive active area, according tovarious embodiments. The β-hydroxybutyrate dehydrogenase and the NADHoxidase may be covalently bonded to a polymer in the ketones-responsiveactive area, and the NAD⁺ may or may not be covalently bonded to apolymer in the ketones-responsive active area. If the NAD⁺ is notcovalently bonded, it may be physically retained within theketones-responsive active area, with a membrane polymer promotingretention of the NAD⁺ within the ketones-responsive active area. Thereis again a 1:1 molar correspondence between the amount of electronstransferred to the working electrode and the amount of β-hydroxybutyrateconverted, thereby providing the basis for ketones detection.

Another enzymatic detection chemistry for ketones may utilizeβ-hydroxybutyrate dehydrogenase (HBDH) to convert β-hydroxybutyrate andNAD⁺ into acetoacetate and NADH, respectively. The electron transfercycle in this case is completed by oxidation of NADH by1,10-phenanthroline-5,6-dione to reform NAD⁺, wherein the1,10-phenanthroline-5,6-dione subsequently transfers electrons to theworking electrode. The 1,10-phenanthroline-5,6-dione may or may not becovalently bonded to a polymer within the ketones-responsive activearea. The β-hydroxybutyrate dehydrogenase may be covalently bonded to apolymer in the ketones-responsive active area, and the NAD⁺ may or maynot be covalently bonded to a polymer in the ketones-responsive activearea. Inclusion of an albumin in the active area may provide asurprising improvement in response stability. A suitable membranepolymer may promote retention of the NAD⁺ within the ketones-responsiveactive area. There is again a 1:1 molar correspondence between theamount of electrons transferred to the working electrode and the amountof β-hydroxybutyrate converted, thereby providing the basis for ketonedetection.

In some embodiments, the analyte sensors may further comprise acreatinine-responsive active area comprising an enzyme system thatoperates in concert to facilitate detection of creatinine. Creatininemay react reversibly and hydrolytically in the presence of creatinineamidohydrolase (CNH) to form creatine. Creatine, in turn, may undergocatalytic hydrolysis in the presence of creatine amidohydrolase (CRH) toform sarcosine. Neither of these reactions produces a flow of electrons(e.g., oxidation or reduction) to provide a basis for electrochemicaldetection of the creatinine. The sarcosine produced via hydrolysis ofcreatine may undergo oxidation in the presence of the oxidized form ofsarcosine oxidase (SOX-ox) to form glycine and formaldehyde, therebygenerating the reduced form of sarcosine oxidase (SOX-red) in theprocess. Hydrogen peroxide also may be generated in the presence ofoxygen. The reduced form of sarcosine oxidase, in turn, may then undergore-oxidation in the presence of the oxidized form of an electrontransfer agent (e.g., an Os(III) complex), thereby producing thecorresponding reduced form of the electron transfer agent (e.g., anOs(II) complex) and delivering a flow of electrons to the workingelectrode.

Oxygen may interfere with the concerted sequence of reactions used todetect creatinine in accordance with the disclosure above. Specifically,the reduced form of sarcosine oxidase may undergo a reaction with oxygento reform the corresponding oxidized form of this enzyme but withoutexchanging electrons with the electron transfer agent. Although theenzymes all remain active when the reaction with oxygen occurs, noelectrons flow to the working electrode. Without being bound by theoryor mechanism, the competing reaction with oxygen is believed to resultfrom kinetic effects. That is, oxidation of the reduced form ofsarcosine oxidase with oxygen is believed to occur faster than doesoxidation promoted by the electron transfer agent. Hydrogen peroxide isalso formed in the presence of the oxygen.

The desired reaction pathway for facilitating detection of creatininemay be encouraged by including an oxygen scavenger in proximity to theenzyme system. Various oxygen scavengers and dispositions thereof may besuitable, including oxidase enzymes such as glucose oxidase. Smallmolecule oxygen scavengers may also be suitable, but they may be fullyconsumed before the sensor lifetime is otherwise fully exhausted.Enzymes, in contrast, may undergo reversible oxidation and reduction,thereby affording a longer sensor lifetime. By discouraging oxidation ofthe reduced form of sarcosine oxidase with oxygen, the slower electronexchange reaction with the electron transfer agent may occur, therebyallowing production of a current at the working electrode. The magnitudeof the current produced is proportional to the amount of creatinine thatwas initially reacted.

The oxygen scavenger used for encouraging the desired reaction may be anoxidase enzyme in any embodiment of the present disclosure. Any oxidaseenzyme may be used to promote oxygen scavenging in proximity to theenzyme system, provided that a suitable substrate for the enzyme is alsopresent, thereby providing a reagent for reacting with the oxygen in thepresence of the oxidase enzyme. Oxidase enzymes that may be suitable foroxygen scavenging in the present disclosure include, but are not limitedto, glucose oxidase, lactate oxidase, xanthine oxidase, and the like.Glucose oxidase may be a particularly desirable oxidase enzyme topromote oxygen scavenging due to the ready availability of glucose invarious bodily fluids. Reaction 1 below shows the enzymatic reactionpromoted by glucose oxidase to afford oxygen clearing.

β-D-glucose+O₂—→D-glucono-1,5-lactone+H₂O₂  Reaction 1

The concentration of available lactate in vivo is lower than that ofglucose, but still sufficient to promote oxygen scavenging.

Oxidase enzymes, such as glucose oxidase, may be positioned in anylocation suitable to promote oxygen scavenging in the analyte sensorsdisclosed herein. Glucose oxidase, for example, may be positioned uponthe sensor tail such that the glucose oxidase is functional and/ornon-functional for promoting glucose detection. When non-functional forpromoting glucose detection, the glucose oxidase may be positioned uponthe sensor tail such that electrons produced during glucose oxidationare precluded from reaching the working electrode, such as throughelectrically isolating the glucose oxidase from the working electrode.

Additional details concerning enzyme systems responsive to creatininemay be found in commonly owned U.S. patent application Ser. No.16/774,835 entitled “Analyte Sensors and Sensing Methods for DetectingCreatinine,” filed on Sep. 25, 2019, and published as U.S. PatentApplication Publication 2020/0241015, the entirety of which isincorporated herein by reference.

In some embodiments, the analyte sensors may comprise alactate-responsive active area comprising a lactate-responsive enzymedisposed upon the sensor tail. Suitable lactate-responsive enzymes mayinclude, for example, lactate oxidase. Lactate oxidase or otherlactate-responsive enzymes may be covalently bonded to a polymercomprising the lactate-responsive active area and exchange electronswith an electron transfer agent (e.g., an osmium (Os)) complex orsimilar transition metal complex), which may also be covalently bondedto the polymer. Suitable electron transfer agents are described infurther detail below. An albumin, such as human serum albumin, may bepresent in the lactate-responsive active area to stabilize the sensorresponse, as described in further detail in commonly owned U.S. PatentApplication Publication 2019/0320947, which is incorporated herein byreference in its entirety. Lactate levels may vary in response tonumerous environmental or physiological factors including, for example,eating, stress, exercise, sepsis or septic shock, infection, hypoxia,presence of cancerous tissue, or the like.

In some embodiments, the analyte sensors may comprise an active arearesponsive to pH. Suitable analyte sensors configured for determining pHare described in commonly owned U.S. Patent Application Publication2020/0060592, which is incorporated herein by reference in its entirety.Such analyte sensors may comprise a sensor tail comprising a firstworking electrode and a second working electrode, wherein a first activearea located upon the first working electrode comprises a substancehaving pH-dependent oxidation-reduction chemistry, and a second activearea located upon the second working electrode comprises a substancehaving oxidation-reduction chemistry that is substantially invariantwith pH. By obtaining a difference between the first signal and thesecond signal, the difference may be correlated to the pH of a fluid towhich the analyte sensor is exposed.

Two different types of active areas may be located upon a single workingelectrode, such as the carbon working electrodes discussed above, andspaced apart from one another. Each active area may have anoxidation-reduction potential, wherein the oxidation-reduction potentialof the first active area is sufficiently separated from theoxidation-reduction potential of the second active area to allowindependent production of a signal from one of the active areas. By wayof non-limiting example, the oxidation-reduction potentials may differby at least about 100 mV, or by at least about 150 mV, or by at leastabout 200 mV. The upper limit of the separation between theoxidation-reduction potentials is dictated by the workingelectrochemical window in vivo. By having the oxidation-reductionpotentials of the two active areas sufficiently separated in magnitudefrom one another, an electrochemical reaction may take place within oneof the two active areas (i.e., within the first active area or thesecond active area) without substantially inducing an electrochemicalreaction within the other active area. Thus, a signal from one of thefirst active area or the second active area may be independentlyproduced at or above its corresponding oxidation-reduction potential(the lower oxidation-reduction potential) but below theoxidation-reduction potential of the other active area. A differentsignal may allow the signal contribution from each analyte to beresolved.

Some or all embodiments of analyte sensors disclosed herein may featureone or more active areas located upon the surface of at least oneworking electrode, where the active areas detect the same or differentanalytes. A membrane may overcoat at least the active area (comprisingan analyte-responsive enzyme), and may further overcoat all or a portionof the working electrode lacking an active area (the exposed orextraneous portion of the working electrode). The membrane may be a masstransport limiting membrane and may be a single layer of membrane, abilayer of two different membrane polymers, or an admixture of twodifferent membrane polymers

An electron transfer agent may be present in any of the active areasdisclosed herein. Suitable electron transfer agents may facilitateconveyance of electrons to the adjacent working electrode after one ormore analytes undergoes an enzymatic oxidation-reduction reaction withinthe corresponding active area, thereby generating an electron flow thatis indicative of the presence of a particular analyte. The amount ofcurrent generated is proportional to the quantity of analyte that ispresent. Depending on the sensor configuration used, the electrontransfer agents in active areas responsive to different analytes may bethe same or different. For example, when two different active areas aredisposed upon the same working electrode, the electron transfer agentwithin each active area may be different (e.g., chemically differentsuch that the electron transfer agents exhibit differentoxidation-reduction potentials). When multiple working electrodes arepresent, the electron transfer agent within each active area may be thesame or different, since each working electrode may be interrogatedseparately.

Suitable electron transfer agents may include electroreducible andelectrooxidizable ions, complexes or molecules (e.g., quinones) havingoxidation-reduction potentials that are a few hundred millivolts aboveor below the oxidation-reduction potential of the standard calomelelectrode (SCE). According to some embodiments, suitable electrontransfer agents may include low-potential osmium complexes, such asthose described in U.S. Pat. Nos. 6,134,461 and 6,605,200, which areincorporated herein by reference in their entirety. Additional examplesof suitable electron transfer agents include those described in U.S.Pat. Nos. 6,736,957, 7,501,053 and 7,754,093, the disclosures of each ofwhich are incorporated herein by reference in their entirety. Othersuitable electron transfer agents may comprise metal compounds orcomplexes of ruthenium, osmium, iron (e.g., polyvinylferrocene orhexacyanoferrate), or cobalt, including metallocene compounds thereof,for example. Suitable ligands for the metal complexes may also include,for example, bidentate or higher denticity ligands such as, for example,bipyridine, biimidazole, phenanthroline, or pyridyl(imidazole). Othersuitable bidentate ligands may include, for example, amino acids, oxalicacid, acetylacetone, diaminoalkanes, or o-diaminoarenes. Any combinationof monodentate, bidentate, tridentate, tetradentate, or higher denticityligands may be present in a metal complex to achieve a full coordinationsphere.

Active areas suitable for detecting any of the analytes disclosed hereinmay comprise a polymer to which the electron transfer agents arecovalently bound. Any of the electron transfer agents disclosed hereinmay comprise suitable functionality to promote covalent bonding to thepolymer within the active areas. Suitable examples of polymer-boundelectron transfer agents may include those described in U.S. Pat. Nos.8,444,834, 8,268,143 and 6,605,201, the disclosures of which areincorporated herein by reference in their entirety. Suitable polymersfor inclusion in the active areas may include, but are not limited to,polyvinylpyridines (e.g., poly(4-vinylpyridine)), polyvinylimidazoles(e.g., poly(l-vinylimidazole)), or any copolymer thereof. Illustrativecopolymers that may be suitable for inclusion in the active areasinclude those containing monomer units such as styrene, acrylamide,methacrylamide, or acrylonitrile, for example. When two or moredifferent active areas are present, the polymer within each active areamay be the same or different.

Covalent bonding of the electron transfer agent to a polymer within anactive area may take place by polymerizing a monomer unit bearing acovalently bonded electron transfer agent, or the electron transferagent may be reacted with the polymer separately after the polymer hasalready been synthesized. A bifunctional spacer may covalently bond theelectron transfer agent to the polymer within the active area, with afirst functional group being reactive with the polymer (e.g., afunctional group capable of quaternizing a pyridine nitrogen atom or animidazole nitrogen atom) and a second functional group being reactivewith the electron transfer agent (e.g., a functional group that isreactive with a ligand coordinating a metal ion).

Similarly, one or more of the enzymes within the active areas may becovalently bonded to a polymer comprising an active area. When an enzymesystem comprising multiple enzymes is present in a given active area,all of the multiple enzymes may be covalently bonded to the polymer insome embodiments, and in other embodiments, only a portion of themultiple enzymes may be covalently bonded to the polymer. For example,one or more enzymes comprising an enzyme system may be covalently bondedto the polymer and at least one enzyme may be non-covalently associatedwith the polymer, such that the non-covalently bonded enzyme isphysically entrained within the polymer. Covalent bonding of theenzyme(s) to the polymer in a given active area may take place via acrosslinker introduced with a suitable crosslinking agent. Suitablecrosslinking agents for reaction with free amino groups in the enzyme(e.g., with the free side chain amine in lysine) may includecrosslinking agents such as, for example, polyethylene glycol diglycidylether (PEGDGE) or other polyepoxides, cyanuric chloride,N-hydroxysuccinimide, imidoesters, epichlorohydrin, or derivatizedvariants thereof. Suitable crosslinking agents for reaction with freecarboxylic acid groups in the enzyme may include, for example,carbodiimides. The crosslinking of the enzyme to the polymer isgenerally intermolecular, but can be intramolecular in some embodiments.In particular embodiments, all of the enzymes within a given active areamay be covalently bonded to a polymer.

The electron transfer agent and/or the enzyme(s) may be associated withthe polymer in an active area through means other than covalent bondingas well. In some embodiments, the electron transfer agent and/or theenzyme(s) may be ionically or coordinatively associated with thepolymer. For example, a charged polymer may be ionically associated withan oppositely charged electron transfer agent or enzyme(s). In stillother embodiments, the electron transfer agent and/or the enzyme(s) maybe physically entrained within the polymer without being bonded thereto.Physically entrained electron transfer agents and/or enzyme(s) may stillsuitably interact with a fluid to promote analyte detection withoutbeing substantially leached from the active areas.

The polymer within the active area may be chosen such that outwarddiffusion of NAD⁺ or another cofactor not covalently bound to thepolymer is limited. Limited outward diffusion of the cofactor maypromote a reasonable sensor lifetime (days to weeks) while stillallowing sufficient inward analyte diffusion to promote detection.

In some embodiments, a stabilizer may be incorporated into the activearea of the analyte sensors described herein to improve thefunctionality of the sensors and achieve desired sensitivity andstability. Such stabilizers may include an antioxidant and/or companionprotein to stabilize the enzyme, for instance. Examples of suitablestabilizers may include, but are not limited to, serum albumin (e.g.,humane or bovine serum albumin or other compatible albumin), catalase,other enzyme antioxidants, and the like, and any combination thereof.The stabilizers may be conjugated or non-conjugated.

In particular embodiments of the present disclosure, the mass transportlimiting membrane overcoating one or more active areas may comprise acrosslinked polyvinylpyridine homopolymer or copolymer. The compositionof the mass transport limiting membrane may be the same or differentwhere the mass transport limiting membrane overcoats active areas ofdiffering types. When the membrane composition varies at two differentlocations, the membrane may comprise a bilayer membrane or a homogeneousadmixture of two different membrane polymers, one of which may be acrosslinked polyvinylpyridine or polyvinylimidazole homopolymer orcopolymer. Suitable techniques for depositing a mass transport limitingmembrane upon the active area may include, for example, spray coating,painting, inkjet printing, screen printing, stenciling, roller coating,dip coating, the like, and any combination thereof. Dip coatingtechniques may be especially desirable for polyvinylpyridine andpolyvinylimidazole polymers and copolymers.

In certain embodiments, the mass transport limiting membrane discussedabove is a membrane composed of crosslinked polymers containingheterocyclic nitrogen groups, such as polymers of polyvinylpyridine andpolyvinylimidazole. Embodiments also include membranes that are made ofa polyurethane, or polyether urethane, or chemically related material,or membranes that are made of silicone, and the like.

In some embodiments, a membrane may be formed by crosslinking in situ apolymer, including those discussed above, modified with a zwitterionicmoiety, a non-pyridine copolymer component, and optionally anothermoiety that is either hydrophilic or hydrophobic, and/or has otherdesirable properties, in a buffer solution (e.g., an alcohol-buffersolution). The modified polymer may be made from a precursor polymercontaining heterocyclic nitrogen groups. For example, a precursorpolymer may be polyvinylpyridine or polyvinylimidazole. Optionally,hydrophilic or hydrophobic modifiers may be used to “fine-tune” thepermeability of the resulting membrane to an analyte of interest.Optional hydrophilic modifiers, such as poly(ethylene glycol), hydroxylor polyhydroxyl modifiers, and the like, and any combinations thereof,may be used to enhance the biocompatibility of the polymer or theresulting membrane.

In some embodiments, the membrane may comprise a compound including, butnot limited to, poly(styrene-co-maleic anhydride), dodecylamine andpoly(propylene glycol)-block-poly(ethylene glycol)-block-poly(propyleneglycol) (2-aminopropyl ether) crosslinked with poly(propyleneglycol)-block-poly(ethylene glycol)-block-poly(propylene glycol)bis(2-aminopropyl ether); poly(N-isopropyl acrylamide); a copolymer ofpoly(ethylene oxide) and poly(propylene oxide); polyvinylpyridine; aderivative of polyvinylpyridine; polyvinylimidazole; a derivative ofpolyvinylimidazole; polyvinylpyrrolidone (PVP), and the like; and anycombination thereof. In some embodiments, the membrane may be comprisedof a polyvinylpyridine-co-styrene polymer, in which a portion of thepyridine nitrogen atoms are functionalized with a non-crosslinkedpoly(ethylene glycol) tail and a portion of the pyridine nitrogen atomsare functionalized with an alkylsulfonic acid group. Other membranecompounds, alone or in combination with any aforementioned membranecompounds, may comprise a suitable copolymer of 4-vinylpyridine andstyrene and an amine-free polyether arm.

The membrane compounds described herein may further be crosslinked withone or more crosslinking agents, including those listed above withreference to the enzyme described herein. For example, suitablecrosslinking agents may include, but are not limited to, polyethyleneglycol diglycidylether (PEGDGE), glycerol triglycidyl ether (Gly3),polydimethylsiloxane diglycidylether (PDMS-DGE), or other polyepoxides,cyanuric chloride, N-hydroxysuccinimide, imidoesters, epichlorohydrin,or derivatized variants thereof, and any combination thereof. Branchedversions with similar terminal chemistry are also suitable for thepresent disclosure. For example, in some embodiments, Formula 1 may becrosslinked with triglycidyl glycerol ether and/or PEDGE and/orpolydimethylsiloxane diglycidylether (PDMS-DGE).

A membrane may be formed in situ by applying an alcohol-buffer solutionof a crosslinker and a modified polymer over the active area and anyadditional compounds included in the active area (e.g., electrontransfer agent) and allowing the solution to cure for about one to twodays or other appropriate time period. The crosslinker-polymer solutionmay be applied over the active area by placing a droplet or droplets ofthe membrane solution on at least the sensor element(s) of the sensortail, by dipping the sensor tail into the membrane solution, by sprayingthe membrane solution on the sensor, by heat pressing or melting themembrane in any sized layer (such as discrete or all encompassing) andeither before or after singulation, vapor deposition of the membrane,powder coating of the membrane, and the like, and any combinationthereof. In order to coat the distal and side edges of the sensor, themembrane material may be applied subsequent to application (e.g.,singulation) of the sensor electronic precursors (e.g., electrodes). Insome embodiments, the analyte sensor is dip-coated following electronicprecursor application to apply one or more membranes. Alternatively, theanalyte sensor could be slot-die coated wherein each side of the analytesensor is coated separately. A membrane applied in the above manner mayhave any of various functions including, but not limited to, masstransport limitation (i.e., reduction or elimination of the flux of oneor more analytes and/or compounds that reach the active area),biocompatibility enhancement, interferent reduction, and the like, andany combination thereof.

Generally, the thickness of the membrane is controlled by theconcentration of the membrane solution, by the number of droplets of themembrane solution applied, by the number of times the sensor is dippedin the membrane solution, by the volume of membrane solution sprayed onthe sensor, and the like, and by any combination of these factors. Insome embodiments, the membrane described herein may have a thicknessranging from about 0.1 μm to about 1000 μm, encompassing any value andsubset therebetween and in which the upper and lower limits areseparable. As stated above, the membrane may overlay one or more activeareas, and in some embodiments, the active areas may have a thickness offrom about 0.1 μm to about 50 μm, encompassing any value and subsettherebetween and in which the upper and lower limits are separable. Insome embodiments, a series of droplets may be applied atop one anotherto achieve the desired thickness of the active area and/or membrane,without substantially increasing the diameter of the applied droplets(i.e., maintaining the desired diameter or range thereof). Each singledroplet, for example, may be applied and then allowed to cool or dry,followed by one or more additional droplets. Active areas and membranemay, but need not be, the same thickness throughout or compositionthroughout.

In some embodiments, the membrane composition for use as a masstransport limiting layer of the present disclosure may comprisepolydimethylsiloxane (PDMS), polydimethylsiloxane diglycidylether(PDMS-DGE), aminopropyl terminated polydimethylsiloxane, and the like,and any combination thereof for use as a leveling agent (e.g., forreducing the contact angle of the membrane composition or active areacomposition). Branched versions with similar terminal chemistry are alsosuitable for the present disclosure. Certain leveling agents mayadditionally be included, such as those found, for example, in U.S. Pat.No. 8,983,568, the disclosure of which is incorporated by referenceherein in its entirety.

In some instances, the membrane may form one or more bonds with theactive area. As used herein, the term “bonds,” and grammatical variantsthereof, refers to any type of an interaction between atoms or moleculesthat allows chemical compounds to form associations with each other,such as, but not limited to, covalent bonds, ionic bonds, dipole-dipoleinteractions, hydrogen bonds, London dispersion forces, and the like,and any combination thereof. For example, in situ polymerization of themembrane can form crosslinks between the polymers of the membrane andthe polymers in the active area. In some embodiments, crosslinking ofthe membrane to the active area facilitates a reduction in theoccurrence of delamination of the membrane from the sensor.

Embodiments disclosed herein include:

A. A method comprising: laser singulating a working electrode comprisingan active area disposed thereon, the active area having ananalyte-responsive enzyme, wherein the resultant laser singulatedworking electrode comprises electrode asperities; and laser planing atleast a portion of the electrode asperities, the laser planing beingrecessed from an edge of the laser singulated working electrode, andthereby resulting in a laser planed working electrode.

B. An analyte sensor comprising: a working electrode comprising anactive area disposed thereon, the active area having ananalyte-responsive enzyme, wherein the working electrode is first lasersingulated, thereby resulting in electrode asperities, and thereafter anedge of the working electrode is laser planed to remove at least aportion of electrode asperities therefrom, thereby resulting in a laserplaned working electrode.

C. A method comprising: laser singulating a working electrode comprisingan active area disposed thereon, the active area having ananalyte-responsive enzyme, wherein the resultant laser singulatedworking electrode comprises electrode asperities; disposing a membraneupon at least a portion of the active area; and laser planing at least aportion of the electrode asperities, the laser planing being recessedfrom an edge of the laser singulated working electrode, and therebyresulting in a laser planed working electrode.

D. An analyte sensor comprising: a laser planed working electrodecomprising an active area disposed thereon and electrode asperitieslaser planed therefrom, the active area comprising an analyte-responsiveenzyme.

E. A method comprising: exposing an analyte sensor to a bodily fluid,the analyte sensor comprising a laser planed working electrodecomprising an active area disposed thereon and electrode asperitieslaser planed therefrom, the active area comprising an analyte-responsiveenzyme.

F. An analyte sensor comprising: a working electrode comprising anactive area disposed thereon, the active area including ananalyte-responsive enzyme; a membrane disposed upon at least a portionof the working electrode comprising the active area; and aninterferent-reactant species incorporated into the analyte sensor.

G. A method comprising: exposing an analyte sensor to a bodily fluid,the analyte sensor comprising: a working electrode comprising an activearea disposed thereon, the active area including an analyte-responsiveenzyme; a membrane disposed upon at least a portion of the workingelectrode comprising the active area; and an interferent-reactantspecies incorporated into the analyte sensor.

H. An analyte sensor comprising: a working electrode comprising anactive area disposed thereon, the active area including ananalyte-responsive enzyme; and a scrubbing electrode, wherein thescrubbing electrode is (1) positioned in a facing relationship to theworking electrode and the working electrode and scrubbing electrode arespatially offset or (2) positioned above the working electrode and ispermeable to an analyte of interest for diffusion of the analyte ofinterest to the active area.

I. A method comprising: exposing an analyte sensor to a bodily fluid,the analyte sensor comprising: a working electrode comprising an activearea disposed thereon, the active area including an analyte-responsiveenzyme; and a scrubbing electrode, wherein the scrubbing electrode is(1) positioned in a facing relationship to the working electrode and theworking electrode and scrubbing electrode are spatially offset or (2)positioned above the working electrode and is permeable to an analyte ofinterest for diffusion of the analyte of interest to the active area.

J. An analyte sensor comprising: a working electrode comprising anactive area disposed thereon, the active area including ananalyte-responsive enzyme; and a scrubbing electrode positioned in afacing relationship to the working electrode, wherein the workingelectrode and scrubbing electrode are spatially offset.

K. A method comprising: exposing an analyte sensor to a bodily fluid,the analyte sensor comprising: a working electrode comprising an activearea disposed thereon, the active area including an analyte-responsiveenzyme; and a scrubbing electrode positioned in a facing relationship tothe working electrode, wherein the working electrode and scrubbingelectrode are spatially offset.

L. An analyte sensor comprising: a working electrode comprising anactive area disposed thereon, the active area including ananalyte-responsive enzyme; and a permeable scrubbing electrodepositioned above the working electrode, wherein the permeable scrubbingelectrode is permeable to an analyte of interest for diffusion of theanalyte of interest to the active area.

M. A method comprising: exposing an analyte sensor to a bodily fluid,the analyte sensor comprising: a working electrode comprising an activearea disposed thereon, the active area including an analyte-responsiveenzyme; and a permeable scrubbing electrode positioned above the workingelectrode, wherein the permeable scrubbing electrode is permeable to ananalyte of interest for diffusion of the analyte of interest to theactive area.

Embodiment A may have one or more of the following additional elementsin any combination:

Element A1: wherein the laser planed working electrode exhibits areduction in interferent signal of an interferent compared to a workingelectrode that has not been planed.

Element A2: wherein the laser planed working electrode exhibits areduction in interferent signal of an interferent compared to a workingelectrode that has not been planed, the reduction in interferent signalof the interferent being greater than about 20%.

Element A3: wherein the laser planed working electrode exhibits areduction in interferent signal of an interferent compared to a workingelectrode that has not been planed, the reduction in interferent signalof the interferent being in the range of about 20% to about 70%,encompassing any value and subset therebetween and in which the upperand lower limits are separable.

Element A4: wherein the laser planed working electrode exhibits areduction in interferent signal of an interferent compared to a workingelectrode that has not been planed, the interferent being ascorbic acid.

Element A5: further comprising removing at least about 5% of a totalarea of the electrode asperities.

Element A6: further comprising removing in the range of about 5% toabout 75% of a total area of the electrode asperities, encompassing anyvalue and subset therebetween and in which the upper and lower limitsare separable.

Element A7: wherein the laser planing comprises a plurality ofsingle-pass laser planing cuts.

Element A8: wherein the laser planing comprises a plurality ofsingle-pass laser planing cuts, the plurality of single-pass laserplaning cuts being at least one of perpendicular to the edge of thelaser singulated working electrode or beveled relative to the edge ofthe laser singulated working electrode.

Element A9: wherein the electrode asperities have a width in the rangeof 1 μm to about 75 μm, encompassing any value and subset therebetweenand in which the upper and lower limits are separable.

Element A10: wherein the electrode asperities have a height of about 1μm to about 50 μm, encompassing any value and subset therebetween and inwhich the upper and lower limits are separable.

Element A11: wherein the active area is comprised of a plurality ofdiscontiguous active areas or a single contiguous active area.

Element A12: wherein the active area is compressed.

Element A13: wherein the active-responsive enzyme is a glucoseresponsive enzyme.

Element A14: wherein a membrane is disposed upon at least a portion ofthe active area.

By way of non-limiting example, exemplary combinations applicable to Ainclude, but are not limited to: A with any one or more or all ofA1-A14, in any combination.

Embodiment B may have one or more of the following additional elementsin any combination:

Element B1: wherein the laser planed working electrode exhibits areduction in interferent signal of an interferent compared to a workingelectrode that has not been planed.

Element B2: wherein the laser planed working electrode exhibits areduction in interferent signal of an interferent compared to a workingelectrode that has not been planed, the reduction in interferent signalof the interferent being greater than about 20%.

Element B3: wherein the laser planed working electrode exhibits areduction in interferent signal of an interferent compared to a workingelectrode that has not been planed, the reduction in interferent signalof the interferent being in the range of about 20% to about 70%,encompassing any value and subset therebetween and in which the upperand lower limits are separable.

Element B4: wherein the laser planed working electrode exhibits areduction in interferent signal of an interferent compared to a workingelectrode that has not been planed, the interferent being ascorbic acid.

Element B5: wherein at least about 5% of a total area of the electrodeasperities is removed.

Element B6: wherein in the range of about 5% to about 75% of a totalarea of the electrode asperities is removed, encompassing any value andsubset therebetween and in which the upper and lower limits areseparable.

Element B7: wherein the electrode asperities have a width in the rangeof 1 μm to about 75 μm, encompassing any value and subset therebetweenand in which the upper and lower limits are separable.

Element B8: wherein the electrode asperities have a height of about 1 μmto about 50 μm, encompassing any value and subset therebetween and inwhich the upper and lower limits are separable.

Element B9: wherein the active area is comprised of a plurality ofdiscontiguous active areas or a single contiguous active area.

Element B10: wherein the active area is compressed.

Element B11: wherein the active-responsive enzyme is a glucoseresponsive enzyme.

Element B12: wherein a membrane is disposed upon at least a portion ofthe active area.

By way of non-limiting example, exemplary combinations applicable to Binclude, but are not limited to: B with any one or more or all ofB1-B12, in any combination.

Embodiment C may have one or more of the following additional elementsin any combination:

Element C1: wherein the laser planed working electrode exhibits areduction in interferent signal of an interferent compared to a workingelectrode that has not been planed.

Element C2: wherein the laser planed working electrode exhibits areduction in interferent signal of an interferent compared to a workingelectrode that has not been planed, the reduction in interferent signalof the interferent being greater than about 20%.

Element C3: wherein the laser planed working electrode exhibits areduction in interferent signal of an interferent compared to a workingelectrode that has not been planed, the reduction in interferent signalof the interferent being in the range of about 20% to about 70%,encompassing any value and subset therebetween and in which the upperand lower limits are separable.

Element C4: wherein the laser planed working electrode exhibits areduction in interferent signal of an interferent compared to a workingelectrode that has not been planed, the interferent being ascorbic acid.

Element C5: further comprising removing at least about 5% of a totalarea of the electrode asperities.

Element C6: further comprising removing in the range of about 5% toabout 75% of a total area of the electrode asperities, encompassing anyvalue and subset therebetween and in which the upper and lower limitsare separable.

Element C7: wherein the laser planing comprises a plurality ofsingle-pass laser planing cuts.

Element C8: wherein the laser planing comprises a plurality ofsingle-pass laser planing cuts, the plurality of single-pass laserplaning cuts being at least one of perpendicular to the edge of thelaser singulated working electrode or beveled relative to the edge ofthe laser singulated working electrode.

Element C9: wherein the electrode asperities have a width in the rangeof 1 μm to about 75 μm, encompassing any value and subset therebetweenand in which the upper and lower limits are separable.

Element C10: wherein the electrode asperities have a height of about 1μm to about 50 μm, encompassing any value and subset therebetween and inwhich the upper and lower limits are separable.

Element C11: wherein the active area is comprised of a plurality ofdiscontiguous active areas or a single contiguous active area.

Element C12: wherein the active area is compressed.

Element C13: wherein the active-responsive enzyme is a glucoseresponsive enzyme.

By way of non-limiting example, exemplary combinations applicable to Cinclude, but are not limited to: C with any one or more or all ofC1-C13, in any combination.

Embodiments D and E may have one or more of the following additionalelements in any combination:

Element D/E1: wherein the laser planed working electrode exhibits areduction in interferent signal of an interferent compared to a workingelectrode that has not been planed.

Element D/E2: wherein the laser planed working electrode exhibits areduction in interferent signal of an interferent compared to a workingelectrode that has not been planed, the reduction in interferent signalof the interferent being greater than about 20%.

Element D/E3: wherein the laser planed working electrode exhibits areduction in interferent signal of an interferent compared to a workingelectrode that has not been planed, the reduction in interferent signalof the interferent being in the range of about 20% to about 70%,encompassing any value and subset therebetween and in which the upperand lower limits are separable.

Element D/E4: wherein the laser planed working electrode exhibits areduction in interferent signal of an interferent compared to a workingelectrode that has not been planed, the interferent being ascorbic acid.

Element D/E5: wherein at least about 5% of a total area of the electrodeasperities is removed.

Element D/E6: wherein in the range of about 5% to about 75% of a totalarea of the electrode asperities is removed, encompassing any value andsubset therebetween and in which the upper and lower limits areseparable.

Element D/E7: wherein the electrode asperities have a width in the rangeof 1 μm to about 75 μm, encompassing any value and subset therebetweenand in which the upper and lower limits are separable.

Element D/E8: wherein the electrode asperities have a height of about 1μm to about 50 μm, encompassing any value and subset therebetween and inwhich the upper and lower limits are separable.

Element D/E9: wherein the active area is comprised of a plurality ofdiscontiguous active areas or a single contiguous active area.

Element D/E10: wherein the active area is compressed.

Element D/E11: wherein the active-responsive enzyme is a glucoseresponsive enzyme.

Element D/E12: wherein a membrane is disposed upon at least a portion ofthe active area.

By way of non-limiting example, exemplary combinations applicable to D/Einclude, but are not limited to: D/E with any one or more or all ofD/E1-D/E12, in any combination.

Embodiments H through M, depending on the configuration of the scrubbingelectrode as described below, may have one or more of the followingadditional elements in any combination:

Element H-M1: wherein the scrubbing electrode is positioned in a facingrelationship to the working electrode and the working electrode andscrubbing electrode are spatially offset, the offset being a thin layerin the range of about 1 μm to about 200 μm; encompassing any value andsubset therebetween and in which the upper and lower limits areseparable

Element H-M2: wherein the scrubbing electrode has a width of about 200μm to about 8000 μm, encompassing any value and subset therebetween andin which the upper and lower limits are separable.

Element H-M3: wherein the scrubbing electrode is positioned in a facingrelationship to the working electrode and the working electrode andscrubbing electrode are spatially offset, and the width of the scrubbingelectrode to the working electrode is in the range of about 2:1 to about50:1, encompassing any value and subset therebetween and in which theupper and lower limits are separable.

Element H-M4: wherein the scrubbing electrode has a potential in therange of about −2000 mV to about +2000 mV, encompassing any value andsubset therebetween and in which the upper and lower limits areseparable.

Element H-M5: wherein the scrubbing electrode is positioned above theworking electrode and is permeable to an analyte of interest fordiffusion of the analyte of interest to the active area, the analyte ofinterest being ascorbic acid.

Element H-M6: wherein the scrubbing electrode is positioned above theworking electrode and is permeable to an analyte of interest fordiffusion of the analyte of interest to the active area, the scrubbingelectrode composed of a carbon nanotube material.

Element H-M7: wherein the scrubbing electrode is positioned above theworking electrode and is permeable to an analyte of interest fordiffusion of the analyte of interest to the active area, the scrubbingelectrode composed of PEDOT:PSS impregnated with a carbon nanotubematerial.

Element H-M8: wherein the scrubbing electrode is positioned above theworking electrode and is permeable to an analyte of interest fordiffusion of the analyte of interest to the active area, the scrubbingelectrode impregnated with an electron transfer agent.

Element H-M9: wherein the scrubbing electrode is positioned above theworking electrode and is permeable to an analyte of interest fordiffusion of the analyte of interest to the active area, the scrubbingelectrode having a thickness of about 0.5 μm to about 100 μm,encompassing any value and subset therebetween and in which the upperand lower limits are separable.

By way of non-limiting example, exemplary combinations applicable to H-Minclude, but are not limited to: H-M with any one or more or all ofH-M1-H-M9, in any combination, depending on the scrubbing electrodeconfiguration (i.e., spatially offset or permeable).

To facilitate a better understanding of the embodiments describedherein, the following examples of various representative embodiments aregiven. In no way should the following examples be read to limit, or todefine, the scope of the invention.

Example 1. In this Example, laser planing was performed on the examplelaser singulated working electrode shown in FIG. 13A. FIG. 13A does notcomprise an active area disposed thereupon. FIG. 13B shows a 3D opticalprofile of a portion of singulated working electrode of FIG. 13A,evaluated along the identified profile width. The 3D optical profile wasobtained using a ZEGAGE™ 3D Optical Profiler, ZYGOO® Corporation(Middlefield, Conn.). As shown in FIG. 13B, the electrode asperities atthe edge of the singulated sensor tail exhibited a height of about 1 μmto about 5 μm.

Laser planing was performed using three single-pass laser linespositioned at the edge of the carbon asperities and made about 5 μm toabout 20 μm, such as about 5 μm to about 10 μm, apart progressivelytoward the midline of the electrode at 10% laser power. In the examplesdescribed herein, a UV laser was used, but it is to be appreciated thatany laser may be used to perform laser planing, without departing fromthe scope of the present disclosure. FIG. 13C is a photograph of theplaned sensor tail, showing the beveled edge of the working electrode ofthe sensor tail. FIG. 13D is a 3D optical profile (obtained aspreviously described) along the identified profile line showing theelectrode asperities removed.

Example 2. In this Example, and with reference to FIG. 14A, a laserplaned carbon working electrode 1400 was prepared in accordance withExample 1, the carbon electrode comprising active areas 1402 dispensedthereupon. The unplaned carbon electrode comprising active areas 1402 isnot shown, but will be referred to as “unplaned, dispensed” electrode.FIG. 14B is a 3D optical profile (obtained as previously described)along the identified profile line showing minimal electrode asperitiesas a result of the planing.

Example 3. A paired-difference test was performed. The unplanedelectrode of FIG. 13A and planed electrode of FIG. 13C having no activearea (“not planed, not dispensed” and “planed, not dispensed,”respectively) were examined with the “unplaned, dispensed” electrode ofExample 2 and the planed electrode of FIG. 14A having multiple sensingspots (“planed, dispensed”) were evaluated in 100 mM PBS at 37° C.separately in 50 mg/dL glucose and 2 mg/dL ascorbic acid. The resultsare provided in Table 1 below, and graphically represented in FIG. 15.

TABLE 1 Iavg (nA) n = 8* Undispensed Dispensed Not Planed Planed Planed(FIG. 13A) (FIG. 13C) Not Planed (FIG. 14A) Glucose ~0 ~0 ~5 ~5 AscorbicAcid ~3 ~2 ~3 ~2 % Δ** ~−30 ~−25 *background corrected; **laser-planedrelative to control

As shown, the paired-different test demonstrates that the laser planedelectrodes demonstrate a reduction in 2 mg/dL of ascorbic acid by about25% to about 30% compared to the unplaned counterparts.

Example 4. Paired-difference tests were performed on the followingprepared laser singulated working electrodes. The unplaned “control”working electrodes comprised active areas of multiple sensing spots. Theelectrodes described as “compressed” comprise the same concentration ofactive area (compressed active area), but the multiple sensing spots arecloser together and/or closer to the tip of the electrode. For example,the pitch between each discontiguous active area (the distance betweenadjacent sensing spots) may be about 50 μm to about 800 such as about 50μm to about 500 encompassing any value and subset therebetween and inwhich the upper and lower limits are separable. As used herein, the term“pitch,” and grammatical variants thereof, refers to the spacing betweenadjacent sensing spots in an active area sensing layer, measured fromthe center of each adjacent sensing spot. Typically, the distal mostactive area is located at least about 200 μm to 300 μm (measured fromthe center of the sensing thereof) from the tip of the working electrode(which may be identical to the tip of the sensor tail) to be locatedmost distally into bodily fluid, but may be in the range of about 50 μmto about 500 encompassing any value and subset therebetween and in whichthe upper and lower limits are separable.

The totality of analyte-responsive enzyme for all samples was the same,whether compressed or not. Laser planing is described with reference tothree separate single-pass laser lines, each a particular distance fromthe edge of the initial unplaned electrode (the “planing scheme”). Forexample, “20-40-60” refers to a first single-pass laser line at 20 μmfrom the edge of the unplaned electrode, a second single-pass laser lineat 40 μm from the edge of the unplaned electrode, and a thirdsingle-pass laser line at 60 μm from the edge of the unplaned electrode.

TABLE 2 FIG. 16A FIG. 16B FIG. 16C FIG. 16D FIG. 16E Compressed? No NoYes Yes Yes Planing Unplaned 20-40-60 Unplaned 20-40-60 15-25-40 Scheme

The electrodes 16A-16E described in Table 2 in some instances werecoated with a mass transport limiting membrane having the thicknessshown in Table 3 below. Paired-difference tests (avg. of n=6/condition)were performed in 100 mM PBS at 37° C. separately in 50 mg/dL glucoseand 2 mg/dL ascorbic acid. The results are provided in Table 3 below.

TABLE 3 Planing? Membrane Thickness % Δ 16A No ~35 μm ~0 16A No ~50 μm~−20 16B 20-40-60 ~35 μm ~−30 16B 20-40-60 ~50 μm ~−45 16C No ~35 μm~−30 16C No ~50 μm ~−50 16D 20-40-60 ~35 μm ~−50 16D 20-40-60 ~50 μm~−65 16E 15-25-40 ~35 μm ~−50 16E 15-25-40 ~50 μm ~−60

As shown in Table 3, the paired-difference tests demonstrate that thelaser planed electrodes may demonstrate a reduction in ascorbic acidsignal interference by greater than about 20% compared to the unplanedcounterparts. Accordingly, the embodiments of the present disclosurepermit at least a reduction in interferent signal, such as ascorbicacid, in the range of greater than about 20%, which may be up to 100%depending on the configuration of the analyte sensor, or such as in therange of about 20% to about 70% or greater, and preferably at leastabout 40% greater, at least about 45% greater, or at least about 50%,encompassing any value and subset therebetween and in which the upperand lower limits are separable. Further, the results indicate that evena relatively small laser planing amount can be effective.

Example 5. In this example, the effectiveness of incorporating anenzymatic interferent-reactant species into an analyte sensor foreliminating or reducing interferent signal at the working electrode wasevaluated. Glucose sensors 1700 having an interferent-reactant layer forreacting with ascorbic acid were prepared, as shown in FIG. 17. Aglucose active area sensing layer 1704 was coated onto the carbonworking electrode 1702 (the carbon electrode disposed upon a substrate(not shown)) in the form of six discontiguous sensing spots andcomprising glucose oxidase sensing chemistry. A diffusion-limitingmembrane 1706 was coated upon the entire working electrode 1702,covering each of the sensing spots 1702. 50 nL of aninterferent-reactant species layer 1708 was coated atop thediffusion-limiting membrane 1706, covering the sensing layer 1704 andany excess (exposed) carbon working electrode 1702. Theinterferent-reactant species layer 1708 comprised ascorbate oxidase (˜25mg/ml) in a matrix of PVI polymer (˜9 mg/ml), PEDGE-400 crosslinker (˜6mg/ml), and albumin stabilizer (˜25 mg/ml) (Solutions were made in 10 mMIVIES buffer, pH 5.5).

Two types of ascorbate oxidase were evaluated, ASO-301 and ASO-311, eachavailable from TOYOBO, headquartered in Osaka, Japan. A thin layer ofcrosslinked outer membrane 1710 was coated atop the entirety of theinterferent-reactant species layer 1708. These sensors are referred toas “GOx/Membrane/Crosslinker/AscOx301” and“GOx/Membrane/Crosslinker/AscOx311,” depending on the ascorbate oxidaseused.

The sensors were tested in 100 mM PBS at a temperature of ˜30° C., a pHof ˜7.5, and a working potential of +40 mV, along with two controls, inquadruplicate, separately in ascorbic acid and glucose. The firstcontrol (“Gox/Membrane Control”) comprised the carbon working electrode1702, sensing spots 1703, and diffusion-limiting membrane 1704 (absentthe interferent-reactant species layer 1708, and the outer membranelayer). The second control (“Gox/Membrane/Crosslinker”) comprised thecarbon working electrode 1702, sensing spots 1704, diffusion-limitingmembrane 1706, and the outer membrane layer 1710 coated thereupon(absent the interferent-reactant species layer 1708). The sensors werecalibrated in ascorbic acid, as shown in FIG. 18, and in 30 mM glucose,as shown in FIG. 19.

As shown, the sensors with the interferent-reactant layer (comprisingascorbate oxidase) show very minimal response to ascorbic acidadditions, as compared to the control sensors both with and without thePVP membrane. Further, the inclusion of the interferent-reactant layerdid not have an appreciable influence on the response to glucose, ascompared to the control sensors both with and without the PVP membrane.Moreover, even if the interferent-reactant layer had affected theglucose sensing, as long as linearity and stability for glucose isretained, any such affect could be easily accounted for. Accordingly,incorporation of an enzymatic interferent-reactant species layer is aviable method to eliminate or reduce signal at the working electrodeattributable to interferents.

Example 6. In this example, the effectiveness of incorporating a metaloxide interferent-reactant species into an analyte sensor foreliminating or reducing interferent signal at the working electrode wasevaluated. Glucose sensors having an interferent-reactant layer forreacting with ascorbic acid were prepared, as shown in FIG. 17. Aglucose active area sensing layer 1702 was coated onto the carbonworking electrode 1702, and the carbon electrode disposed upon asubstrate (not shown). The active area sensing layer 1702 was in theform of six discontiguous sensing spots comprising glucose oxidasesensing chemistry. The active area had a total area of ˜0.1 mm². Theworking electrodes comprising the sensing spots were dipped in adiffusion-limiting membrane comprising either a control polymercomposition or an experimental polymer composition including MnO2(Catalog #217646, available from SIGMA-ALDRICH, headquartered in St.Louis, Mo.). The control and experimental diffusion-limiting membraneswere allowed to cure.

The sensors were beaker tested in 100 mM PBS at a temperature of ˜30°C., along with two controls, in quadruplicate, separately in 1 mg/mlascorbic acid and 5 mM glucose. The sensor current results are shown inTable 5.

TABLE 5 Sensor Current (nA) 1 mg/ml Ascorbic % Ascorbic Acid 5 mMGlucose Acid Interference Control ~10 ~2 ~20% Experimental ~11 ~1  ~8%

As shown, the experimental sensors comprising the interferent-reactantspecies within the diffusion-limiting membrane show reduced ascorbicacid interference. Accordingly, incorporation of a metal oxideinterferent-reactant species is a viable method to eliminate or reducesignal at the working electrode attributable to interferents.

Example 7. In this example, the effectiveness of incorporating ascrubbing electrode into an analyte sensor for eliminating or reducinginterferent signal at the working electrode was evaluated. A glucosesensor was prepared by applying a glucose sensing active area of glucoseoxidase chemistry to a working electrode. The working electrode wasapproximately 200 μm in width. A scrubbing electrode was incorporated byapplying a layer of adhesive to create a thin layer of about 50 Thescrubbing electrode was approximately 2500 μm in width. Nodiffusion-limiting membrane was incorporated into the sensor.

The sensor was beaker tested in 1 mM glucose in 100 mM PBS at atemperature of ˜30° C. FIG. 20 shows the current for both the workingelectrode 2002 and the scrubbing electrode 2004. As shown, the workingelectrode 2002 maintains a substantially stable glucose response and thescrubbing electrode 2004 exhibits no response to glucose, as expectedbecause it comprises no glucose sensing chemistry, for upwards of twoweeks, even absent the diffusion-limiting membrane. Accordingly, thediffusion-limiting function of the membrane may be achieved using ascrubbing electrode.

Example 8. In this example, the effectiveness of incorporating ascrubbing electrode into an analyte sensor for eliminating or reducinginterferent signal at the working electrode was evaluated. The glucosesensor comprising the scrubbing electrode of Example 7 was tested in thepresence of glucose and ascorbic acid, with potential applied or removedfrom the scrubbing electrode. When potential was applied to either theworking electrode or the scrubbing electrode, the potential was +40 mV.As shown in FIG. 21A, the sensor was beaker tested in 100 mM PBS at atemperature of ˜30° C. After approximately 24 hours, 250 μM of glucosewas added at 2102 and the working electrode function 2104 and scrubbingelectrode function 2106 were observed. As shown, the working electrode2104 peaks to a steady state upon detecting the glucose and thescrubbing electrode 2106 remains essentially unaffected. Afterapproximately 25 hours, 2 mg/dL (˜115 μM) of ascorbic acid was added2108 and, as shown, the working electrode 2104 response remained steady(detecting glucose), and the scrubbing electrode 2106 response currentincreased instantaneously (detecting ascorbic acid 2108). Thereafter,the potential of the scrubbing electrode 2106 was turned off (labeled2110) and back on again (labeled 2110), and the comparative increasebetween these actions of the glucose signal may be attributed toascorbic acid 2102. FIG. 21B shows the sensor after approximately 18days, demonstrating its stability over at least this time period—at21142 mg/ml of ascorbic acid was added and at 2116 the scrubbingelectrode 2106 was again turned off. Accordingly, it is apparent thatthe scrubbing electrode is effective in removing ascorbic acid fromaccessing the working electrode.

Example 9. In this example, the effectiveness of incorporating ascrubbing electrode into an analyte sensor for eliminating or reducinginterferent signal at the working electrode was evaluated. Two glucosesensors comprising a scrubbing electrode were prepared according toExample 7, each having a different carbon ink type and different screenprinting locations. The scrubbing electrodes were prepared by StevenLabel Corporation (Santa Fe Springs, Calif.) (labeled “C1” in FIG. 22,the thicker line) and in-house (labeled “C2” in FIG. 22, the thinnerline). The commercial composition of the carbon inks are different(e.g., different carbon particles, different binders, and/or differentratio of carbon to binder). Moreover, the location of screen printingwas different, due to for example proprietary printing processes,temperatures, curing times, and the like. The two different sensors werebeaker tested in 100 mM PBS at a temperature of ˜30° C. in 2.0 mg/dLascorbic acid, the ascorbic acid added at approximately 0.8 hours andcorresponding to the second spike in current. The sensor currents ofeach of the scrubbing electrodes are shown in FIG. 22 in response tovarious applied potentials in mV (labeled as +40, +100, +200, +300, and+400, and “Off” in which no potential was applied), and it is evidentthat the scrubbing electrode composition material, location, andpotential applied to the scrubbing electrode can influence its scrubbingefficiency. Accordingly, the scrubbing electrode may be optimized inview of the interferent of interest and/or its concentration in a bodilyfluid, and the like, and any combination thereof.

Example 10. In this example, the effectiveness of incorporating ananalyte-permeable scrubbing electrode into an analyte sensor foreliminating or reducing interferent signal at the working electrode wasevaluated. Glucose sensors 2300 comprising a carbon nanotubeanalyte-permeable electrode were prepared as shown in FIG. 23. Theworking electrode 2304 was screen printed onto a plastic substrate 2302with surrounding well(s) 2306 to allow for deposited solutions ofadditional components of the sensor to be tested. The well 2306 isrepresented as the “well boundary” portions 2306 a,b of FIG. 23. Thiswell configuration, and variants thereof, may be used in the embodimentsof the present disclosure, as described above. An active area 2308 ofketone sensing chemistry was automated liquid dispensed into the well2306 and atop (in contact with) the working electrode 2304. While thisexample utilized ketone sensing chemistry, it is to be understood thatother analytes, such as glucose, lactate, creatinine, ethanol, and thelike, and multiple combinations thereof may be employed as describedherein without departing from the scope of the present disclosure. Theactive area 2308 covered a portion of the working electrode, but excess(exposed) working electrode 2304 a remained present, shown to the leftand right of active area 2308. Thereafter, an initial diffusion-limitingmembrane 2310 a (e.g., of 10Q5 or other membrane polymer, such as thosedescribed herein) was deposited (e.g., manually or automated deposition)into the well atop the active area 2308 and the excess working electrodeportions 2304 a. A carbon nanotube analyte-permeable scrubbing electrode2311 was deposited into the well atop the initial membrane 2310 a,followed by a dip-coating (or other deposition manner) of the entiresensor in a second diffusion-limiting coating (e.g., a membrane polymer,such as those described herein). The initial and second membranecoatings 2310 a and 2310 b, respectively may be the same or differentcomposition dependent at least upon the analyte(s) being detected,without departing from the scope of the present disclosure.

As shown in FIG. 24, ketone sensors comprising the carbon nanotubeanalyte-permeable scrubbing electrode as shown in FIG. 23 and describedhereinabove were beaker tested in 100 mM PBS. After approximately 1 hour(˜1.3 hours), 5 mg/dL of ascorbic acid was added and the workingelectrode 2404 (thicker line) and scrubbing electrode 2406 (thinnerline) were observed. As shown, the addition of the ascorbic acidresulted in interferent signal from the working electrode. Afterapplying a +40 mV potential to the scrubbing electrode, the interferentsignal of the working electrode decreased by ˜85%. The scrubbingelectrode was disconnected, and the interferent signal on the baseelectrode returned to previous levels. The scrubbing electrode was againconnected and the potential applied was adjusted to +40, +200, and +600mV, with modest improvements in scrubbing efficiency at higherpotentials. While not shown, in addition to ketone, it was observed thatvarious other analytes of interest, including glucose and betahydroxybutyrate, readily diffused through the scrubbing electrode togenerate signal at the underlying working electrode.

Accordingly, the present disclosure provides analyte sensors are formonitoring various analytes in vivo. The analyte sensors may featureenhancements to address signals obtained from interferent species. Someanalyte sensors may comprise a working electrode comprising an activearea disposed thereon and electrode asperities laser planed therefrom.Some analyte sensors may comprise an interferent-reactant speciesincorporated therewith. Some analyte sensors may comprise an interferentscrubbing electrode. Combinations of these enhancements may additionallybe employed.

Unless otherwise indicated, all numbers expressing quantities and thelike in the present specification and associated claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the specification and attached claims are approximationsthat may vary depending upon the desired properties sought to beobtained by the embodiments of the present invention. At the very least,and not as an attempt to limit the application of the doctrine ofequivalents to the scope of the claim, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques.

One or more illustrative embodiments incorporating various features arepresented herein. Not all features of a physical implementation aredescribed or shown in this application for the sake of clarity. It isunderstood that in the development of a physical embodimentincorporating the embodiments of the present invention, numerousimplementation-specific decisions must be made to achieve thedeveloper's goals, such as compliance with system-related,business-related, government-related and other constraints, which varyby implementation and from time to time. While a developer's effortsmight be time-consuming, such efforts would be, nevertheless, a routineundertaking for those of ordinary skill in the art and having benefit ofthis disclosure.

While various systems, tools and methods are described herein in termsof “comprising” various components or steps, the systems, tools andmethods can also “consist essentially of” or “consist of” the variouscomponents and steps.

As used herein, the phrase “at least one of” preceding a series ofitems, with the terms “and” or “or” to separate any of the items,modifies the list as a whole, rather than each member of the list (i.e.,each item). The phrase “at least one of” allows a meaning that includesat least one of any one of the items, and/or at least one of anycombination of the items, and/or at least one of each of the items. Byway of example, the phrases “at least one of A, B, and C” or “at leastone of A, B, or C” each refer to only A, only B, or only C; anycombination of A, B, and C; and/or at least one of each of A, B, and C.

Therefore, the disclosed systems, tools and methods are well adapted toattain the ends and advantages mentioned as well as those that areinherent therein. The particular embodiments disclosed above areillustrative only, as the teachings of the present disclosure may bemodified and practiced in different but equivalent manners apparent tothose skilled in the art having the benefit of the teachings herein.Furthermore, no limitations are intended to the details of constructionor design herein shown, other than as described in the claims below. Itis therefore evident that the particular illustrative embodimentsdisclosed above may be altered, combined, or modified and all suchvariations are considered within the scope of the present disclosure.The systems, tools and methods illustratively disclosed herein maysuitably be practiced in the absence of any element that is notspecifically disclosed herein and/or any optional element disclosedherein. While systems, tools and methods are described in terms of“comprising,” “containing,” or “including” various components or steps,the systems, tools and methods can also “consist essentially of” or“consist of” the various components and steps. All numbers and rangesdisclosed above may vary by some amount. Whenever a numerical range witha lower limit and an upper limit is disclosed, any number and anyincluded range falling within the range is specifically disclosed. Inparticular, every range of values (of the form, “from about a to aboutb,” or, equivalently, “from approximately a to b,” or, equivalently,“from approximately a-b”) disclosed herein is to be understood to setforth every number and range encompassed within the broader range ofvalues. Also, the terms in the claims have their plain, ordinary meaningunless otherwise explicitly and clearly defined by the patentee.Moreover, the indefinite articles “a” or “an,” as used in the claims,are defined herein to mean one or more than one of the elements that itintroduces. If there is any conflict in the usages of a word or term inthis specification and one or more patent or other documents that may beincorporated herein by reference, the definitions that are consistentwith this specification should be adopted.

What is claimed is:
 1. An analyte sensor comprising: a working electrodecomprising an active area disposed thereon, the active area including ananalyte-responsive enzyme; and a scrubbing electrode, wherein thescrubbing electrode is (1) positioned in a facing relationship to theworking electrode and the working electrode and scrubbing electrode arespatially offset or (2) positioned above the working electrode and ispermeable to an analyte of interest for diffusion of the analyte ofinterest to the active area.
 2. The analyte sensor of claim 1, whereinthe scrubbing electrode is positioned in a facing relationship to theworking electrode and the working electrode and scrubbing electrode arespatially offset, the offset being a thin layer in the range of about 1μm to about 200 μm.
 3. The analyte sensor of claim 1, wherein thescrubbing electrode has a width of about 200 μm to about 8000 μm.
 4. Theanalyte sensor of claim 1, wherein the scrubbing electrode is positionedin a facing relationship to the working electrode and the workingelectrode and scrubbing electrode are spatially offset, and the width ofthe scrubbing electrode to the working electrode is in the range ofabout 2:1 to about 50:1.
 5. The analyte sensor of claim 1, wherein thescrubbing electrode has a potential in the range of about −2000 mV toabout +2000 mV.
 6. The analyte sensor of claim 1, wherein the scrubbingelectrode is positioned above the working electrode and is permeable toan analyte of interest for diffusion of the analyte of interest to theactive area, the analyte of interest being ascorbic acid.
 7. The analytesensor of claim 1, wherein the scrubbing electrode is positioned abovethe working electrode and is permeable to an analyte of interest fordiffusion of the analyte of interest to the active area, the scrubbingelectrode composed of a carbon nanotube material.
 8. The analyte sensorof claim 1, wherein the scrubbing electrode is positioned above theworking electrode and is permeable to an analyte of interest fordiffusion of the analyte of interest to the active area, the scrubbingelectrode composed of PEDOT:PSS impregnated with a carbon nanotubematerial.
 9. The analyte sensor of claim 1, wherein the scrubbingelectrode is positioned above the working electrode and is permeable toan analyte of interest for diffusion of the analyte of interest to theactive area, the scrubbing electrode impregnated with an electrontransfer agent.
 10. The analyte sensor of claim 1, wherein the scrubbingelectrode is positioned above the working electrode and is permeable toan analyte of interest for diffusion of the analyte of interest to theactive area, the scrubbing electrode having a thickness of about 0.5 μmto about 100 μm.
 11. A method comprising: exposing an analyte sensor toa bodily fluid, the analyte sensor comprising: a working electrodecomprising an active area disposed thereon, the active area including ananalyte-responsive enzyme; and a scrubbing electrode, wherein thescrubbing electrode is (1) positioned in a facing relationship to theworking electrode and the working electrode and scrubbing electrode arespatially offset or (2) positioned above the working electrode and ispermeable to an analyte of interest for diffusion of the analyte ofinterest to the active area.
 12. The method of claim 11, wherein thescrubbing electrode is positioned in a facing relationship to theworking electrode and the working electrode and scrubbing electrode arespatially offset, the offset being a thin layer in the range of about 1μm to about 200 μm.
 13. The method of claim 11, wherein the scrubbingelectrode has a width of about 200 μm to about 8000 μm.
 14. The methodof claim 11, wherein the scrubbing electrode is positioned in a facingrelationship to the working electrode and the working electrode andscrubbing electrode are spatially offset, and the width of the scrubbingelectrode to the working electrode is in the range of about 2:1 to about50:1.
 15. The method of claim 11, wherein the scrubbing electrode has apotential in the range of about −2000 mV to about +2000 mV.
 16. Themethod of claim 11, wherein the scrubbing electrode is positioned abovethe working electrode and is permeable to an analyte of interest fordiffusion of the analyte of interest to the active area, the analyte ofinterest being ascorbic acid.
 17. The method of claim 11, wherein thescrubbing electrode is positioned above the working electrode and ispermeable to an analyte of interest for diffusion of the analyte ofinterest to the active area, the scrubbing electrode composed of acarbon nanotube material.
 18. The method of claim 11, wherein thescrubbing electrode is positioned above the working electrode and ispermeable to an analyte of interest for diffusion of the analyte ofinterest to the active area, the scrubbing electrode composed ofPEDOT:PSS impregnated with a carbon nanotube material.
 19. The method ofclaim 11, wherein the scrubbing electrode is positioned above theworking electrode and is permeable to an analyte of interest fordiffusion of the analyte of interest to the active area, the scrubbingelectrode impregnated with an electron transfer agent.
 20. The method ofclaim 11, wherein the scrubbing electrode is positioned above theworking electrode and is permeable to an analyte of interest fordiffusion of the analyte of interest to the active area, the scrubbingelectrode having a thickness of about 0.5 μm to about 100 μm.